CN1594567A - Immune tbid gene, encoded protein and use thereof - Google Patents
Immune tbid gene, encoded protein and use thereof Download PDFInfo
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- CN1594567A CN1594567A CN 200410026316 CN200410026316A CN1594567A CN 1594567 A CN1594567 A CN 1594567A CN 200410026316 CN200410026316 CN 200410026316 CN 200410026316 A CN200410026316 A CN 200410026316A CN 1594567 A CN1594567 A CN 1594567A
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Abstract
The invention concerns a tbid gene, protein coding by tbid gene and uses of said gene and protein in tumor drug preparation. In the invention, single chain antibody e23sFv gene which can idiocraticly identify and bind HER-2 positive tumor cell is used to construct a tbid gene with PEA transmembrane structural domain and active truncated bid gene (tbid)which has strong specificity,high activity ,low immunogenicity,sustainable execution on tumor. The invention constructs secretion type immunological target gene. after gene is expressed and secreted, its can produce effector molecule to kill tumor cell. The gene therapy strategy possessing simultaneously the cell immunity and humoral immunity character has obvious advantages compared with other strategy. Immunological tbid gene the invention constructed is safe, highly effective and continuous effect. The invention is hoped to provide a novel method for HER-2 tumor target therapy.
Description
Technical field
The present invention relates to the gene therapy of biological technical field, be specifically related to gene engineering method structure immune tbid gene (we are its called after Immuno-tbid), said gene encoded protein and described gene and albumen are used for the medicine of oncotherapy in preparation application.
Background technology
Along with the continuous appearance of medical science new technology, the surgical intervention effect of primary tumor, particularly early diagnosis person is significantly improved.But be far apart the transfer of organ when tumour cell after, an especially general transferrer, conventional therapy such as chemotherapy, radiotherapy, operative treatment etc. all can not reach the control to tumour.Secretion expression's targeting proteins inducing apoptosis of tumour cell and kill the thinking of tumour, some are efficient with instructing, the appearance of therapy of tumor new departure of low toxicity.
HER-2 is a kind of EGF-R ELISA by the proto-oncogene coding, express at tumour cell camber such as mammary cancer, ovarian cancers, it is a kind of generally acknowledged tumor markers, and, much studies show that, HER-2 male Prognosis in Breast Cancer is often very poor, and tumour shows the ability of very strong transfer, does not also have effective medicine and means at present.By to the antigenic specific recognition of HER-2, be expected under the not hurtful situation of normal cell, realize specific killing to malignant tumour.
Bid (BH3 Interacting Death Agonist; BH3 interaction pro apoptotic protein) is a member of the BH3 of the Bcl-2 family subfamily of discovery in 1996, has pro-apoptosis bioactivity.Bid albumen only contains the BH3 structural domain, interacts cell death inducing when it crosses expression in cell by it and Bcl-2 and Bax.Its title is also come therefrom.
The pro-apoptosis bioactivity of Bid mainly depends on the shearing of Caspase-8.When apoptotic signal stimulates, the Caspase-8 activation also cuts away Bid albumen n end fragment, produce Bid truncate segment and insert to plastosome from cytoplasm, cause multiple and apoptosis associated molecule by discharging in the plastosome, thereby realize regulating apoptosis by Cytc approach and other plastosome apoptosis mechanism.
The Bid truncate be it is advantageous that as the tumor-killing effector molecule, it is the key molecule that connects apoptosis inherence and external approach, by inherent approach, amplify the apoptotic signal of external approach, activate several apoptosis pathway simultaneously, cause cascade reaction, make a plurality of apoptosis effect molecule (caspase3,6,7 and AIF) play a role simultaneously.
Summary of the invention
The novel molecular that the objective of the invention is to make up a kind of high specificity, activity is high, immunogenicity is low, can continue killing tumor cell provides a kind of immune tbid gene, said gene encoded protein and described gene and albumen to be used for the application of the medicine of oncotherapy in preparation.
Technical solution of the present invention is:
Immune tbid gene, it has sequence table＜400〉sequence of one of 1-2.
By the said gene encoded protein, it has sequence table＜400〉sequence of one of 3-4.
Said gene is used for the application of the medicine of oncotherapy in preparation.
Above-mentioned albumen is used for the application of the medicine of oncotherapy in preparation.
The present invention has used can specific recognition and in conjunction with single-chain antibody e23sFv gene (Chen SY, the et al.Nature 1997 of HER-2 positive tumor cell; 385:78-80), change membrane structure territory and active truncation type bid gene (tbid) fusion with PEA.Combine antibody like this, simultaneously to the specific recognition function of tumour cell and the killing ability rapidly and efficiently of active tbid pair cell.The present invention makes up secretor type immunity target gene, produce the effector molecule that to discern specifically with killing tumor cell behind the expression-secretion, no matter be to use direct knurl body of recombinant virus and intramuscular injection, or by feeding back the lymphocyte of genetic modification, or protokaryon, the eukaryotic expression product of gene are gone in patient's body by muscle, knurl body and intravenous injection, all can in killing tumor cell, produce immunosurveillance.This strategies in gene therapy that has cellular immunization and humoral immunization characteristics concurrently is compared with other strategy, has remarkable advantages.
The constructed immune tbid gene of the present invention has following characteristics: the security---owing to merge long antibody and PEA sequence at the N end, thereby before the transfer process neutralization of this fusion rotein after secretion enter tumour cell, do not have the pro-apoptosis bioactivity of tbid owing to can not correctly fold.In addition, the HER-2 positive tumor cell that the immune tbid effector molecule passes through former of antibodies specific ground identification and shifts, and normal tissue cell is not discerned and lethal effect.In addition, immune tbid mainly is made of humanized antibody and people's oneself protein, use repeatedly can not bring out to produce neutralizing antibody in the body, and the reaction that do not cause inflammation during cell generation apoptosis, so the toxic side effect for the treatment of is less.Find in the experiment that the function of adorned lymphocyte own is normal, feeding back this cell can the continuous release effect protein.Finding in the antitumor application of animal model does not have toxicity to its hetero-organization except that HER-2 positive tumor tissue yet; high efficiency-Bid is a signal transduction molecule important in the apoptosis path, in cell, import the tbid molecule of activity form, not only can activate the apoptosis effect of caspases path, the apoptosis induction molecule that can also promote AIF etc. not rely on caspases discharges from plastosome, even caspases is suppressed the generation that also can start apoptosis, make its lethal effect more direct, and efficient is higher to tumour cell; But persistence---long-term expression behind the immune tbid gene transfectional cell, secretion enters blood circulation, and body is carried out secular immunosurveillance.
The present invention is expected to provide a kind of new tool for the targeted therapy of HER-2 positive tumor.
Description of drawings
Fig. 1 is that the agarose gel electrophoresis of total length people bid gene is identified figure.
Fig. 2 is that the agarose gel electrophoresis of truncated-type human bid gene (tbid61 and tbid76) is identified figure.
Fig. 3 is that the agarose gel electrophoresis of pcDNA3-PEA-tbid61 and pcDNA3-PEA-tbid76 plasmid is identified figure.
Fig. 4 is that the agarose gel electrophoresis of pCMV-Immuno-tbid61 or pCMV-Immuno-tbid76 plasmid is identified figure.
Fig. 5 behaves after tbid61 and the tbid76 gene transfection, the metamorphosis figure (* 400) of HeLa cell under the fluorescent microscope.
Fig. 6 behaves after tbid61 and the tbid76 gene transfection, two mark indirect IF staining figure of cell.
Fig. 7 behaves after PEA-tbid61 and the PEA-tbid76 gene transfection, two mark indirect IF staining figure of cell.
Fig. 8 is the graphic representation that cell growth condition changes behind transfection PEA-tbid61 and the PEA-tbid76 gene.
After Fig. 9 is transfection PEA-tbid61 and PEA-tbid76 gene, the painted flow cytometry figure of cell Annexin V.
Figure 10 shows Immuno-tbid61 and the distribution plan of Immuno-tbid76 albumen in transfectional cell for immunofluorescence.
Figure 11 is that the western blot of stably excreting Immuno-tbid61 and the proteic cells and supernatant of Immuno-tbid76 identifies figure.
Figure 12 is the histogram of cell counting result after different cell conditioned medium and the SKBR-3 cell cultures.
Figure 13 contains the specificity histogram relatively that the proteic supernatant pair cell of Immuno-tbid61 kills and wounds.
Figure 14 is in the antitumor applied research of Immuno-tbid61 gene, the graphic representation that gross tumor volume changes.
Figure 15 is in the antitumor applied research of Immuno-tbid61 gene, the graphic representation of laboratory animal lifetime.
Embodiment
1. the structure of immune tbid gene
The immune tbid gene that the present invention makes up comprises three parts, be respectively the single-chain antibody (e23sFv) of antitumor surface antigen HER-2, have the commentaries on classics membrane structure territory of the Pseudomonas aeruginosa extracellular toxin (PEA) that changes the film function and truncation type Bid (tBid) with cell death inducing function.
(1) clone of truncation type bid gene (tbid)
1. the clone of total length bid gene
Extract total RNA of human lymphoma Jurkat cell, cDNA is synthesized in reverse transcription.In the thin-walled tube of PCR special use, add reverse transcription product 2 μ L, 10 * PCR buffer, 5 μ L, 50mM MgCl successively
21.5 μ L, upstream and downstream primer (1E, 2XS) each 1 μ L, 10mM dNTP 1 μ L, Taq archaeal dna polymerase 0.5 μ L and H
2O 38 μ L carry out the PCR reaction.The PCR reaction conditions is: 96 ℃ of sex change 30 seconds; Annealed 30 seconds for 55 ℃; 72 ℃ were extended 45 seconds; Carry out 20 circulations altogether.Amplify the gene fragment bid (accompanying drawing 1) of 588bp.It is cloned into carrier pcDNA3---called after pcDNA3-bid, through sequencing, bid gene fragment order and the sequence among the GenBank that the result obtains are in full accord.
1E：?5′-TTTGAATTCATGGACTGTGAGGTCAACAAC-3′
2XS：5′-TTTTCTAGAGTCGACTCAGTCCATCCCATTTCTGGC-3′
2. the brachymemma of bid gene
In the thin-walled tube of PCR special use, add pcDNA3-bid plasmid 1 μ L, 10 * PCR buffer5 μ L, 50mM MgCl successively
21.5 μ L, upstream and downstream primer (2XS and 3-61E or 2XS and 4-76E) each 1 μ L, 10mM dNTP 1 μ L, Taq archaeal dna polymerase 0.5 μ L and H
2O 39 μ L carry out the PCR reaction, the tbid76 gene (accompanying drawing 2) of the tbid61 gene of amplification coding 61-195aa and coding 76-195aa.The PCR reaction conditions is: 96 ℃ of sex change 30 seconds; Annealed 45 seconds for 55 ℃; 72 ℃ were extended 45 seconds; Carry out 20 circulations altogether.Gene fragment is cloned into respectively in the pcDNA3 carrier, cuts through enzyme and identifies and the order-checking confirmation.
3-61E：5′-TTTGAATTCAT?G?GGCAACCGCAGCAGCCACTCC-3′
4-76E：5′-TTTGAAATGTTCATGTCTGAAAGTCAAGAAGACATC-3′
(2) clone of PEA-tbid61 and PEA-76 fusion gene
Clone on the successful basis at truncation type bid gene (tbid61 and tbid76), change the gene segment in membrane structure territory (280-364aa) with the part of Pseudomonas aeruginosa extracellular toxin PEA, and linearizing pcDNA3 carrier carries out ligation, obtain recombinant plasmid pcDNA3-PEA-tbid61 and pcDNA3-PEA-tbid76, enzyme is cut and is identified that collection of illustrative plates is shown in (accompanying drawing 3).
(3) clone of Immuno-tbid fusion gene
With truncation type bid gene (tbid61 and tbid76) with existing linearizing plasmid pCMV-e23sFv-PEAII (253-364)-X is connected in this chamber, obtain including the carrier for expression of eukaryon of Immuno-tbid61 or Immuno-tbid76 fusion gene, respectively called after pCMV-Immuno-tbid61 or pCMV-Immuno-tbid76.(accompanying drawing 4)
2.tbid the active checking of gene and expression product thereof
Tbid61 and tbid76 gene clone are gone into fluorescent expression vector pIRES2-EGFP, successfully construct pIRES2-EGFP-tbid61 and pIRES2-EGFP-tbid76 plasmid.With the method for liposome, respectively pIRES2-EGFP, pIRES2-EGFP-tbid61 and pIRES2-EGFP-tbid76 are transfected into the HeLa cell.Fluorescence microscope, the cell well-grown of empty carrier transfection group, pIRES2-EGFP-tbid61 and pIRES2-EGFP-tbid76 cells transfected, 12h cavity occurs in the visible parts of fine born of the same parents after transfection, cell volume generation pyknosis, weaken and disperse to the cell fluorescence of 24h pyknosis, many cells have not had profile (accompanying drawing 5) clearly.The result shows, after tbid61 and tbid76 gene transfection enter cell, causes the opening of necrocytosis program.
Wrap up pcDNA3, pcDNA3-tbid61 and pcDNA3-tbid76 transfection HeLa cell respectively with liposome, the anti-people Cytc of rabbit antibody with Cy3 labelled goat anti-people bid antibody and FITC mark behind the 20h carries out the indirect immunofluorescence detection, the result of fluorescence microscope shows, the cell of pcDNA3-tbid61 and pcDNA3-tbid76 transfection group, the fluorescent mark of cytochrome c be transferred to nuclear around, be ring bunch shape and exist, prompting tBid can impel cytochrome c to be discharged into (accompanying drawing 6) in the tenuigenin from plastosome.
3.PEA-tbid61 and PEA-tbid76 fusion gene and the active checking of expression product thereof
(1) PEA-tbid61 and PEA-tbid76 are in intracellular expression
With liposome pcDNA3, pcDNA3-PEA-tbid61 and pcDNA3-PEA-tbid76 transfection HeLa cell, use the fluorescent dye of the anti-people bid of Cy3 labelled goat antibodymediated immunity behind the 20h, the result of fluorescence microscope shows, a small amount of endogenic bid albumen is arranged in the cell of pcDNA3 transfection group, and the cell after pcDNA3-PEA-tbid61 and the pcDNA3-PEA-tbid76 transfection is painted darker, and the illustration purpose gene is great expression (accompanying drawing 7) in cell.
(2) influence of PEA-tbid61 and PEA-tbid76 gene transfection cell growth
PEA-tbid61 and PEA-tbid76 gene transfection HeLa cell, take a sample in different time points, carry out the experiment of tetrazolium bromide (MTT) colorimetric, this experiment is the important method that detects cell survival and growth, in certain cell count scope, the amount (OD490nm value) that the bluish voilet crystallisate forms is directly proportional with the survivaling cell number.Absorbance value and the time of 490nm are figure, the variation (accompanying drawing 8) of cell growth condition behind the demonstration transfection goal gene.The result shows, compares with control group, and the expression of PEA-tbid61 and PEA-tbid76 fusion gene all can suppress the growth of HeLa cell.
(3) the apoptotic generation of the induced expression of PEA-tbid61 and PEA-tbid76
1. the release of the expression trigger cell pigment c of PEA-tbid61 and PEA-tbid76
Use liposome pcDNA3, pcDNA3-PEA-tbid61 and pcDNA3-PEA-tbid76 transfection HeLa cell, carry out indirect IF staining with the anti-people Cytc of the rabbit of FITC mark antibody behind the 20h, the result of fluorescence microscope shows, compare with the pcDNA3 transfection group, after pcDNA3-PEA-tbid61 and the pcDNA3-PEA-tbid76 transfection, the fluorescent mark of cytochrome c be transferred to nuclear around, being ring bunch shape exists, and typical cell shrinkage and pyknosis appear, the nuclear structure havoc, the prompting cytochrome c is discharged into (accompanying drawing 7) in the tenuigenin from plastosome.
PEA-tbid61 and PEA-tbid76 fusion rotein cause phosphatidylserine and turn up
Apoptosis is early stage, and surface of cell membrane phosphatidylserine can occur from the cytolemma turning inside-out, is exposed to the cytolemma outside, and can combine and be detected this moment with Annexin V reagent.It is the important method of identification of cell apoptosis that Annexin V detects.Behind liposome pcDNA3, pcDNA3-PEA-tbid61 and the pcDNA3-PEA-tbid76 transfection SKBR-3 cell 12h, with Annexin V-FITC and PI dyeing, found that, compare with control group, the cell of pcDNA3-PEA-tbid61 and pcDNA3-PEA-tbid76 transfection group presents the tangible positive, positive rate is respectively: 10.9% and 9.2%, illustrate that the expression of fusion gene PEA-tbid61 and PEA-tbid76 can cause apoptotic generation (accompanying drawing 9).
3. the Change of Ultrastructure of cell after PEA-tbid61 and the PEA-tbid76 genetic expression
With liposome pcDNA3, pcDNA3-PEA-tbid61 and pcDNA3-PEA-tbid76 transfection HeLa cell, after the fixing and dyeing, with electron microscope observation transfectional cell ultrastructure.Found that, cell after pcDNA3-PEA-tbid61 and the pcDNA3-PEA-tbid76 transfection presents typical apoptotic cell feature: disappear as the surface of cell membrane microvillus, nuclear membrane and cytolemma are complete, the surface of cell membrane blebbing, chromosome condensation and be gathered in the nuclear membrane periphery.And non-transfected cells or transfection empty carrier cellular form are normal.
4.Immuno-tbid the checking of the outer killing activity of the tumour metamer of gene and expression product thereof
(1) foundation of Immuno-tbid61 and Immuno-tbid76 secretor type HeLa clone
With pCMV-e23sFv-PEA-tbid61 and pCMV-e23sFv-PEA-tbid76 with liposome after, transfection HeLa cell, carry out immunocytochemical stain behind the 48h, can be observed Immuno-tbid61 and Immuno-tbid76 albumen mainly is distributed in endoplasmic reticulum and the golgi body, be the secreting, expressing pattern, and Immuno-tbid61 and Immuno-tbid76 albumen high expression level are to the HeLa cell of HER-2 feminine gender have no adverse effects (accompanying drawing 10).
With the HeLa cell after the transfection,, obtain positive colony after 3 weeks, enlarged culturing with 800 μ g/ml G418 screening.It is the HeLa cells and supernatant that collection is built, carrying out Western blot after desalination also concentrates detects, the proteic existence of Immuno-tbid in the culture supernatant be can detect, cell and energy continuous release Immuno-tbid61 and the proteic HeLa clone of Immuno-tbid76 that screening obtains confirmed thus.(accompanying drawing 11)
The cell that screening is obtained carries out cell counting at different time, and drafting cell growth curve, find that secretion Immuno-tbid61 and the proteic HeLa cell of Immuno-tbid76 have and the similar growth curve of untransfected HeLa cell, show transfection Immuno-tbid61 and Immuno-tbid76 gene after cell can normal growth.
(2) Immuno-tbid61 and Immuno-tbid76 albumen are to the specific killing effect of HER-2 positive tumor cell
It is the HeLa cells and supernatant that collection is built, and is used for the cultivation of the tumour cell of different sources, and research Immuno-tbid61 and Immuno-tbid76 albumen are to the killing activity of tumour cell.
At first, relatively Immuno-tbid61 and Immuno-tbid76 albumen are to the positive breast cancer cell of HER-2---SKBR-3 lethal effect different.Cultivate the SKBR-3 cell to contain the proteic supernatant of Immuno-tbid61 and Immuno-tbid76, carry out cell counting in different time points.The result shows that Immuno-tbid61 and Immuno-tbid76 albumen all can effectively kill and wound the HER-2 positive tumor cell, but stronger with the proteic lethal effect of Immuno-tbid61, and kill rate and incubation time have dependency.(accompanying drawing 12)
Cultivate the Hep2 and the ECV-304 cell of HER-2 male SKBR-3 and SKOV-3 cell and HER-2 feminine gender to contain the proteic supernatant respectively of Immuno-tbid61, whether the checking lethal effect has target.Culturing cell changes liquid every day, and counts, and calculates the kill rate of HeLa cell conditioned medium to the HER-2 positive cell.Found that the growth of HER-2 positive tumor cell obviously is suppressed, and negative tumour cell---Hep2 and normal cell---the ECV-304 growth of HER-2 is unaffected substantially.Show that fully the killing activity of Immuno-tbid61 albumen pair cell is (accompanying drawing 13) with target.
4.Immuno-tbid61 and the anti-tumor in vivo of Immuno-tbid76 gene and expression product thereof is used
The immunohistochemical staining result of nude mice tissue paraffin section de shows, Immuno-tbid61 albumen only optionally is distributed in the tumor tissues of treatment group nude mice, its hetero-organization after one's own heart, in the liver, spleen, lung, kidney, and in the tumor tissues of control group, only detect the expression of a small amount of endogenous bid.Simultaneously, have only that TUNEL dyeing is positive in the tumor tissues of experimental group, show that Immuno-tbid61 not only can specific identification and be incorporated into HER-2 male tumor tissues, also kill tumour effectively by causing apoptosis of tumor cells, do not kill and wound with other normal tissue cells and secretion is produced the proteic muscle tissue of Immuno-tbid61, its hetero-organization is not had toxic side effect.
Claims (4)
1, immune tbid gene, it has sequence table＜400〉sequence of one of 1-2.
2, by the albumen of the genes encoding of claim 1, it has sequence table＜400〉sequence of one of 3-4.
3, gene as claimed in claim 1 is used for the application of the medicine of oncotherapy in preparation.
4, albumen as claimed in claim 2 is used for the application of the medicine of oncotherapy in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838435A (en) * | 2022-10-28 | 2023-03-24 | 中国人民解放军空军军医大学 | Cell apoptosis-related molecule recombinant immune coupling protein and preparation method and application thereof |
| CN117568405A (en) * | 2023-11-14 | 2024-02-20 | 武汉凯德维斯生物技术有限公司 | Oncolytic adenovirus recombinant vector, construction method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838435A (en) * | 2022-10-28 | 2023-03-24 | 中国人民解放军空军军医大学 | Cell apoptosis-related molecule recombinant immune coupling protein and preparation method and application thereof |
| CN115838435B (en) * | 2022-10-28 | 2023-10-03 | 中国人民解放军空军军医大学 | Cell scorch related molecule recombinant immunoconjugate protein, and preparation method and application thereof |
| CN117568405A (en) * | 2023-11-14 | 2024-02-20 | 武汉凯德维斯生物技术有限公司 | Oncolytic adenovirus recombinant vector, construction method and application thereof |
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