CN1258742A - Recombined toxin adenovirus mutant for treating tumor caused by tumor suppressor p53 defect - Google Patents
Recombined toxin adenovirus mutant for treating tumor caused by tumor suppressor p53 defect Download PDFInfo
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- CN1258742A CN1258742A CN 98124026 CN98124026A CN1258742A CN 1258742 A CN1258742 A CN 1258742A CN 98124026 CN98124026 CN 98124026 CN 98124026 A CN98124026 A CN 98124026A CN 1258742 A CN1258742 A CN 1258742A
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Abstract
The present invention features that plasmid pVC45F, plasmid pGL3-control, plasmid pABS.4, and adenovirus plasmid pBHG11, plasmid pUC18, adenovirus mutant dL 1520 etc. are processed through a series of intermediate processes to obtain genetic expression plasmid and other intermediate product. Finally plasmid pBHG-PE and plasmid pAd delta E1b transfected 293 cells are obtained. Adenovirus mutant containing recombinant toxin gene is screened from the formed plaque, identified through PCR process and named as CBC-015. The adenovirus mutant may be used to further develop high-efficiency new antineoplastic medicine for the gene treatment of P53 defect tumor.
Description
The present invention relates to the recombined toxin adenovirus mutant of a kind of novel tool specific treatment tumor caused by tumor suppressor p 53 defect in the genetically engineered.
Tumour is the common frdquently encountered disease of serious harm human health and people's life, and China's annual cancer neopathy number surpassed for 1,600,000 (cancer patients's total number of persons surpasses 1,000 ten thousand).The positive excess cardiovascular and cerebrovascular disease of crossing of cancer form lethal first reason into the mankind.
The sudden change and the disappearance of caused by tumor suppressor p 53 usually takes place in the tumour of most of types of people, all has the change of p53 as 70% cancer, 60% skin carcinoma, 65% colorectal carcinoma etc.And bad luck is, in most of the cases, tumour with caused by tumor suppressor p 53 functional defect is usually insensitive to radiotherapy or chemotherapy, that is to say, have the caused by tumor suppressor p 53 functional defect the tumour patient substantial loss possibility of further treatment, thereby development is at the tumor treatment method of the caused by tumor suppressor p 53 defect particularly important that just seems.
Use a kind of New-type adenovirus mutant (dk1520 of modern genetic engineering technique construction, also claim ONYX-015), lacked the Elb district of coding 55KD albumen (Elb 55K) in the adenoviral gene group, just because of this disappearance, make this mutant adenovirus physical efficiency in the cell of p53 functional defect, carry out self-replacation, and in normal cell, can not carry out self-replacation.That is to say that after in the tumour of this adenoviral mutants injection human P 53 functional defect, this mutant adenovirus physical efficiency is duplicated, bred in tumour cell, thereby finally cause tumour cell cracking or apoptosis and dead, and normal cell and tissue are also unaffected.
But human body is a very complicated organism, and cancer is a kind of especially complicated disease.Any simple medicine is no matter how powerful its function is, and its effect is still limited.Treatment for cancer is entering the epoch of a complex therapy, and the single-gene treatment plan of tumour is just replaced by the polygene treatment plan.Going to treat p53 defective tumour with simple adenoviral mutants dL1520 no doubt has certain curative effect, but its curative effect still can be subjected to restriction to a certain degree.
" attack with poison mould " is exactly the first-selected principle that treatment comprises the various chronic diseases of cancer since ancient times.In modern modality of cancer treatment, toxin (comprises Pseudomonas aeruginosa extracellular toxin [Pesudomonasexotoxin, PE], diphtheria toxin gene [Diphtheria toxin, DT], staphylococcus intracellular toxin A[Staphylococcal enterotoxin A, SEA] and Ricin [Ricin] etc.) also be the active drug of using always.Yet because mycin itself lacks target, its toxic side effect is bigger, so its application has been subjected to the restriction of certain degree.
Purpose of the present invention: lack target in order to overcome the toxitherapy cancer, shortcoming and the present situation of improving unsatisfactory curative effect in the single-gene treatment plan that toxic side effect is big, the present invention combines the two advantage of adenoviral mutants dL1520 and toxin gene, is built into a kind of novel recombined toxin adenovirus mutant with specific treatment tumor caused by tumor suppressor p 53 defect.Called after CBC-015.
Major technique content: owing to treat the unsatisfactory curative effect of p53 functional defect tumour with simple adenoviral mutants dL1520, the present invention has designed the E3 district (the E3 district of deleted adenovirus does not influence the copy function of adenovirus) that toxin gene is inserted adenovirus hominis mutant dL1520, like this, not only kept the adenoviral mutants dL1520 function of self-replacation in the tumour cell of p53 functional defect specifically, and the expression product that makes toxin gene only acts on the tumour cell of p53 functional defect specifically, thereby improved the target of detoxifying function effectively, reduced the toxic side effect of toxin.
This novel recombined toxin adenovirus mutant (called after CBC～015) that the two advantage of adenoviral mutants dL1520 and toxin gene is combined and be built into, can further be developed as the more powerful anti-cancer agent of a kind of efficient, be used for p53 defective genetic treatment of tumor.
The principal feature of the recombined toxin adenovirus mutant of specific treatment tumor caused by tumor suppressor p 53 defect is: (face building process as follows) plasmid pVC45F (A) reclaims the fragment (a) that comprises the PE gene through the restriction endonuclease double digestion; Simultaneously, plasmid pGL3-control (B) obtains carrier pGL3-Δ Luc (b) behind the restriction endonuclease double digestion; With (b) with (a) couple together, thereby be built into PE gene expression plasmid pGL3-PE (c); This expression plasmid obtains PE genetic expression unit (d) through the restriction endonuclease double digestion; Plasmid pABS.4 (C) is through double digestion, obtain carrier pABS.4 (e), (e) is connected with (d) again, connecting product is PE genetic expression intermediate carrier pABS-PE (f), (f) cut through the restriction endonuclease enzyme, obtain PE gene and kanamycin gene and merge fragment (g); Adenoviral plasmid pB-HG11 (D) obtains carrier pBHG11 (h) after enzyme is cut; (h) is connected with (g), from connect product, filters out recombinant plasmid, interstitial granules pBHG-PE (i) in the called after adenovirus; Adenoviral mutants dL1520 (F) cuts through the restriction endonuclease enzyme, reclaims the gland virus dna fragmentation (j) of disappearance E1b; Plasmid pUC18 (E), the carrier pUC18 (k) that behind double digestion, obtains; (k) is connected with (j), filter out recombinant plasmid from connecting after product, called after adenoviral plasmid pAd Δ Elb (l), with (l) and (i) cotransfection 293 cells (13), from the plaque that forms, filter out the adenoviral mutants (14) that contains recombinant toxin gene, identify recombined toxin adenovirus mutant (Z) with PCR.
Above-mentioned plasmid pVC45F (A) can comprise pseudomonas aeruginosa toxin gene or diphtheria toxin gene or staphylococcus intracellular toxin A or Ricin gene.
Plasmid pGL3-control (B) contains reporter gene---luciferase gene, the eukaryotic gene expression regulating and controlling sequence: simian virus 40 (being SV40) gene promoter, SV40 enhanser and SV40 add poly (A) (being polyadenylic acid) signal.
The structural representation of New-type adenovirus mutant CBC-015 is seen accompanying drawing 5, and this figure is the note of accompanying drawing 2.Relevant english abbreviation is described as follows:
1) Ad5:adenovirus type 5,5 type adenovirus.
2) I mu ≈ 360 bP; Bp (base pairs): base pair.
3) ITR:inverted terminal repeat, terminal repeat; Ela:early
Region l a, early transcription district la; Elb:early region lb, the early transcription district
Lb; E3:early region 3, early transcription district are 3.
4) SV40:simian virus 40, simian virus 40; Poly (A): polyadenylic
Acid, polyadenylic acid.
5) ATG; The transcription initiation codon.
The building process of New-type adenovirus mutant CBC-015 (is example with the PE gene) synoptic diagram is referring to accompanying drawing 6, and this figure is the note of accompanying drawing 1
Containing Pseudomonas aeruginosa extracellular toxin [Pesudomonas exotoxin, PE] the plasmid pVC45F (A) of gene, carry out double digestion (1) with restriction endonuclease Nco I and Spe I, reclaim the fragment that comprises the PE gene (a) of 1.8kb (kb:kilobase does base); Simultaneously, plasmid pGL3-control (B) [this plasmid contains reporter gene---luciferase (lu-ciferase, luc) gene, eukaryotic gene expression regulating and controlling sequence SV40 (seeing notes) gene promoter, SV40 enhanser and SV40 add poly (A) signal] carry out the carrier segments PGL3-Δ luc (b) that double digestion (2) reclaims the disappearance luc gene of 3.6kb with nucleic acid restriction endonuclease Nco I and Xba I, then carrier segments pGL3-Δ luc is connected (3) with the PE gene fragment with the T4 dna ligase, after connecting the product transformed into escherichia coli, filter out recombinant plasmid, be the PE gene expression plasmid, called after pGL3-PE (e).PE gene expression plasmid pGL3-PE carries out double digestion (4) with restriction endonuclease Sma I and BamH I, reclaims the PE genetic expression unit (d) of 2.6kb; Simultaneously, plasmid pABS.4 (c) carries out double digestion (5) with Ec1136 II and BamH I, resulting carrier pABS.4 (e) is connected (6) with PE genetic expression unit (d) with the T4 dna ligase, after connecting the product transformed into escherichia coli, filter out recombinant plasmid, called after intermediate carrier pABS-PE (f).Intermediate carrier pABS-PE (f) is after restriction endonuclease Pdc I enzyme is cut (7), reclaim PE gene and the kanamycin gene syzygy fragment (g) of 3.9kb, simultaneously, adenoviral plasmid pBHG11 (D) is resulting carrier pBHG11 (h) and PE gene and kanamycin gene syzygy fragment (g) after restriction endonuclease Pac I enzyme is cut (8), connect (9) with the T4 dna ligase, after connecting product conversion large intestine bar enzyme, filter out recombinant plasmid, interstitial granules pBHG-PE (I) in the called after adenovirus; After adenoviral mutants dL1520 (F) cuts (II) with restriction endonuclease Sph I enzyme, reclaim the spring viral dna fragment (j) of the disappearance Elb of 2.8kb, this fragment includes the various elements that specificity is duplicated in p53 defective tumour in the adenovirus.Simultaneously, plasmid pUC18 (E) resulting carrier pUC18 (k) behind restriction endonuclease Sma I and Sph I double digestion (10) is connected (12) with the fragment (j) of 2.8kb with the T4 dna ligase, after connecting the product transformed into escherichia coli, filter out recombinant plasmid, called after adenoviral plasmid pAd/Elb (1).
With interstitial granules pBHG-PE (i) cotransfection 293 cells (13) (people's proembryo renal cancer cell line in adenoviral plasmid pAd Δ E1b (I) and the adenovirus, be the host cell strain of transfection adenovirus), from the plaque that forms, filter out the adenoviral mutants (14) that contains recombinant toxin gene, identify recombined toxin adenovirus mutant (Z) with PCR (polymerase chain reaction, polymerase chain reaction) method.This recombinant toxin gland virus mutant promptly is the recombined toxin adenovirus mutant that can duplicate in the tumour cell of p53 functional defect specifically.
Positively effect of the present invention: 1. overcome toxin itself shortage target in the modern modality of cancer treatment, the shortcoming that toxic side effect is big.2. replaced the single-gene treatment plan, this treatment plan has not only kept the function of dL1520 self-replacation in the tumour cell of p53 functional defect, and thereby the expression product that the makes toxin gene tumour cell that only acts on the p53 functional defect specifically improved the target of detoxifying function effectively, reduced the toxic side effect of toxin.3. the two advantage of adenoviral mutants dL1520 and toxin gene is combined, can further be developed as the more powerful anti-cancer agent of a kind of efficient.
Accompanying drawing and drawing explanation:
The building process synoptic diagram of Fig. 1 CBC-015
Structural representation Fig. 3 of Fig. 2 CBC-015, the restricted zymogram of Fig. 4 plasmid
The note of annotated map 6 accompanying drawings 1 of Fig. 5 accompanying drawing 2
Sequence number explanation among the figure; (A) plasmid pVC45F; (B) plasmid pGL3-control; (C) plasmid pABS.4; (D) adenoviral plasmid pBHG11; (E) plasmid pUC18; (F) adenoviral mutants dL1520;
(a) PE gene; (b) carrier pGL3-Δ Iuc; (c) PE gene expression plasmid pGL3-PE; (d) PE genetic expression unit; (e) carrier pABS.4; (f) intermediate carrier pABS-PE; (g) PE gene and kanamycin gene merge fragment; (h) carrier pBHGH; (i) interstitial granules in the adenovirus; (j) the adenovirus DNA fragment of disappearance Elb; (k) carrier pUC18; (1) adenoviral plasmid pAd Δ Elb; (Z) PCR identifies recombined toxin adenovirus mutant.
(1) restriction endonuclease Nco I and Spe I double digestion; (2) nucleic acid restriction endonuclease Nco I/Xba I double digestion; (3) connect; (4) restriction endonuclease Sma I/BamHI double digestion; (5) restriction endonuclease Ecll36 II/BamH I double digestion; (6) connect; (7) restriction endonuclease PacI enzyme is cut; (8) restriction endonuclease pac I enzyme is cut; (9) connect; (10) restriction endonuclease Sma I/Sph I double digestion; (11) restriction endonuclease Sph I enzyme is cut; (12) connect; (13) rotaring redyeing 293 cell; (14) select to contain the adenoviral mutants of the mould plain gene of recombinating from plaque; (15) map unit (map units, i.e. mu); (16) Ad5,5 type adenovirus; (17) disappearance (6.3-9.8mu); (18) disappearance (77.5-86.2) mu; (19) the terminal repetition series of ITR; (20) Ela early transcription district la; (21) Elb early transcription district lb; (22) E3 early transcription district 3; (23) ATG transcription initiation password; (24) transcriptional orientation; (25) SV40 gene promoter; (26) toxin gene; (27) SV40 adds poly (A) signal; (28) SV40 enhanser.
Embodiment: see CBC-015 building process in Fig. 2 and the specification sheets.
Get plasmid (A), (B), (C), (D), (E), (F) each 5 μ g, each process is handled respectively in (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12) in Fig. 1.Realize intermediate product (a) and (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), from plaque, screen the adenoviral mutants that contains recombinant toxin gene with final product (i) with (l) behind cotransfection 293 cells, identify recombinant toxin adenopathy mutant by PCR (polymerase chain reaction) method.
Claims (3)
1. adenoviral mutants for the treatment of tumor caused by tumor suppressor p 53 defect is characterized in that: plasmid pVC45F (A) reclaims the fragment (a) that comprises the PE gene behind the restriction endonuclease double digestion; Simultaneously, plasmid pGL3-control (B) obtains carrier pGL3-Δ Luc (b) behind the restriction endonuclease double digestion; With (b) with (a) couple together, thereby be built into PE gene expression plasmid pGL3-PE (c); This expression plasmid is through restriction endonuclease, and double digestion obtains PE genetic expression unit (d); Plasmid pABS.4 (C) obtains carrier pABS.4 (e) through double digestion; (e) is connected with (d), connecting product is PE genetic expression intermediate carrier called after pABS-PE (f) again; (f) cut through the restriction endonuclease enzyme, obtain PE gene and kanamycin gene and merge fragment (g); Adenoviral plasmid pBHG11 (D) obtains carrier pBHG11 (h) after enzyme is cut, (h) is connected with (g), filters out recombinant plasmid, interstitial granules pBHG-PE (i) in the called after adenovirus from connect product; Adenoviral mutants dL1520 (F) cuts through the restriction endonuclease enzyme, reclaims the adenovirus DNA fragment (j) of disappearance E1b; The carrier pUC18 (k) that plasmid pUC18 (E) obtains behind double digestion; (k) is connected with (j), from connect after product, filters out recombinant plasmid, called after adenoviral plasmid pAd Δ Elb (l); With (l) and (i) cotransfection 293 cells (13), from the plaque that forms, filter out the adenoviral mutants (14) that contains recombinant toxin gene, identify recombined toxin adenovirus mutant (Z) with PCR.
2. by the said adenoviral mutants of claim 1, it is characterized in that plasmid pVC45F (A) can comprise pseudomonas aeruginosa extracellular toxin gene or diphtheria toxin gene or staphylococcus intracellular toxin A or Ricin gene.
3. by claim 1,2 said adenoviral mutants, its feature: be that plasmid pGL3-control (B) contains reporter gene---luciferase gene, eukaryotic gene expression regulating and controlling sequence simian virus 40 (being SV40) gene promoter, SV40 enhanser and SV40 add poly (A) (being polyadenylic acid) signal.
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| CN 98124026 CN1258742A (en) | 1998-12-25 | 1998-12-25 | Recombined toxin adenovirus mutant for treating tumor caused by tumor suppressor p53 defect |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003006640A1 (en) * | 2001-07-12 | 2003-01-23 | Qijun Qian | A specific proliferation in tumour cell which can express antioncogene with high efficiency and the use of it |
| CN102051358B (en) * | 2009-11-10 | 2012-08-22 | 复旦大学 | Mosaic tumor specific gene expression regulation kit and application thereof |
| CN1520303B (en) * | 2001-03-27 | 2013-04-24 | 纽约大学 | Turnor therapy with alphavirus-based and high affinity laminin receptor-targeted vectors |
-
1998
- 1998-12-25 CN CN 98124026 patent/CN1258742A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1520303B (en) * | 2001-03-27 | 2013-04-24 | 纽约大学 | Turnor therapy with alphavirus-based and high affinity laminin receptor-targeted vectors |
| WO2003006640A1 (en) * | 2001-07-12 | 2003-01-23 | Qijun Qian | A specific proliferation in tumour cell which can express antioncogene with high efficiency and the use of it |
| US7491525B2 (en) | 2001-07-12 | 2009-02-17 | Qijun Qian | Specific proliferation in tumour cell which can express antioncogene with high efficiency and the use of it |
| CN102051358B (en) * | 2009-11-10 | 2012-08-22 | 复旦大学 | Mosaic tumor specific gene expression regulation kit and application thereof |
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