CN1254719A - Defective adenovirus and its building-up method - Google Patents
Defective adenovirus and its building-up method Download PDFInfo
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- CN1254719A CN1254719A CN99124030A CN99124030A CN1254719A CN 1254719 A CN1254719 A CN 1254719A CN 99124030 A CN99124030 A CN 99124030A CN 99124030 A CN99124030 A CN 99124030A CN 1254719 A CN1254719 A CN 1254719A
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Abstract
The present invention discloses a defective adenovirus as a new type mutation of E1b55Kda protein gene and its building method. By deletion mutation and termination codon, the E1b55Kda protein function of said adhenovirus carrier is deleted and the E1a, E1b, 19Kda and other adenovirus protein functions are retained. As adenovirus E1a and other virus proteins have anticancer action, said defective adenovirus carrier system can be used to treat many tumors.
Description
The present invention relates to life science, is a kind of defective adenoviral vector system and constructing plan thereof for the treatment of tumour.
Malignant tumour seriously jeopardizes human life and health.At present to the conventional treatment of malignant tumour still for operation and put, chemotherapy, this conventional treatment is still not very good to thumping majority tumor treatment curative effect, for the thumping majority chemotherapeutics, its therapeutic index is still lower, and promptly its therapeutic dose and toxicity dose are comparatively approaching.So this treatment plan is usually with tangible toxic action, comprise life-threatening bone marrow depression etc., therefore, research selectively killing tumour cell and not influence Normocellular treatment plan extremely important to oncotherapy, the treatment plan of this selectively killing tumour cell mainly depends on the specific marker of tumour cell, and its curative effect is decided by also whether strictness is limited in the tumour cell this tumor marker.
In recent years, viral protein on these tumour cells is as the specific target target of immunotherapy, but because in tumour generation and evolution, for escaping the immunity monitoring, the a lot of variation takes place in tumour cell, expresses and the downward modulation of angtigen presentation ability as a lot of tumour cell MHC 1 genoids, lacks the second signal that immunocyte stimulates, thereby effectively activating cells poison T cell is killed tumour cell, therefore immunotherapy curative effect clinically and very undesirable.
The object of the present invention is to provide a kind of defective adenoviral of E1b 55Kda protein gene novel mutation and the novel method of structure thereof.
Gene therapy is a kind of new method of the treatment plan of the malignant tumour of rising in recent years.Two kinds of its gene transfection method partitivirus method and non-viral methods, viral method adopt retrovirus, adenovirus, adeno-associated virus, simple exanthema virus and vaccinia virus usually.Retroviral have higher transfection efficiency external, but virus titer is on the low side, and transfection efficiency is lower in vivo, and can only infect the cell of division stage, has the karyomit(e) of being integrated into simultaneously, has the shortcoming of the possibility of canceration.Adeno-associated virus has the ability of transfection division and resting cell, endurable expression.In contrast, non-viral method comprises methods such as liposome and particle gun, and its rotaring redyeing gene expression time is shorter, and transfection efficiency is lower.Adenovirus is the most commonly encountered diseases poisonous carrier of present therapy of tumor, has been widely used in the human body gene treatment plan, has the characteristics of easy production and purifying, and it reaches external all effectively transfection division and resting cell, non-carcinogenesis in vivo.Some viral protein of adenovirus has antitumor action, have as E1a HER-2/neu overexpression tumour is played restraining effect, comprise downward modulation HER-2/neu genetic expression, reduce tumorigenicity, suppress metastases, the multiple effects such as chemosensitivity of inducing apoptosis of tumour cell and enhancing tumour.Its E1 of adenovirus system (comprising E1a and E1b) disappearance commonly used at present, thus make this adenovirus lack the E1a antineoplastic action.
Defective adenoviral of the present invention is E1b 55Kda protein gene excalation and is lacking position insertion terminator codon, the 2809-3329bp disappearance, dna fragmentation TAATGAGTAACTAA is inserted at this disappearance position, contains two terminator codons in this dna fragmentation, is respectively TAA and TGA.
Defective adenoviral of the present invention can be formed mixture with chemotherapeutic agent such as cis-platinum, 5-fluor-uracil ametycin etc., biotoxin such as snake venom toxin, monoclonal antibody such as anti-liver cancer cell antibody etc., and it is as the antitumor drug better effects if.
Defective adenoviral construction process of the present invention is two adenovirus carrier transfections to be given can produce the cell of reorganization, make the 2809-3329bp disappearance of an E1b 55Kda protein gene in two adenovirus carriers, insert terminator codon at its disappearance position, promptly insert dna fragmentation TAATGAGTAACTAA, containing two terminator codons in this dna fragmentation, is respectively TAA and TGA.
Human adenovirus has 6 different subgenus, is divided into A, B, C, D, E and F.They are also inequality to close preferendum, tumorigenicity and the history of disease of host cell.Below with adenovirus C subgenus 5 type Ad5 as illustration.
Adenovirus system is made up of two carriers, and a carrier provides the left arm part of adenovirus, and another carrier provides right arm, these two carriers have the homologous recombination district of 500nt at least, yet its transfectional cell produces recombinant adenovirus, and carrier system plasmid pXC1 contains wild-type Ad5 left arm.PBHG10 provides shortage E3 district Ad5 right arm or pBHGE3 that Ad5 right arm (containing the E3 district) is provided.
The present invention near inserting the connecting joint two ends in the position primer of synthetic polymerase chain reaction be
CTG?GCC?AAT?ACC?AAC?CTT?A
ATA?TGA?GCT?CAC?AAT?GCT?TC
The present invention transforms the people embryo retina cell strain 911 that human embryonic kidney cell's strain 293 or E1 transform with this defective adenoviral transfection to E1, can make effectively to have antitumor adenovirus.
The present invention has following beneficial effect
1. the invention provides a kind of defective adenoviral of E1b 55Kda protein gene novel mutation, prove that through experimentation on animals this adenovirus can be used for treating kinds of tumors.
2. the invention provides a kind of construction process of defective adenoviral of E1b 55Kda protein gene novel mutation.This method can be used for making up novel defective adenoviral, and this method is easily grasped for operator, and can be used for making up multiple novel defective adenoviral, for the gene therapy disease has been set up good basis.
3. experimentation on animals proves this recombinant adenovirus energy selectively killing tumour cell and does not influence Normocellular treatment, lays the foundation for being used for the human tumor treatment from now on.
Embodiment:
Example one, adenovirus E 1 b protein gene excalation reach the structure that inserts the terminator codon carrier in the disappearance district
The pXC.1 carrier is purchased in Canadian Microbix Biosystem Inc. (Toronto), and pXC.1 contains 5 type adenoviral sequence bp22-5790.Using restriction endonuclease Bgl II cuts this carrier in 3329bp, remake partially digested with restriction endonuclease Hind III then, reclaim the 9372bp dna fragmentation, using e. coli dna polymerase I Klenow fragment mends flat to 3 ' recessed end, be disappearance 2809bp-3329bp zone in the pXC.1 carrier (concrete grammar is referring to the molecular cloning experiment guide, and scientific publication 1992 is published).
Synthetic two DNA oligonucleotide fragments are done a connecting joint, and its DNA oligonucleotide sequence is
01?TAATGAGTAACTAA
02?TTAGTTACTCATTA
Each 0.1 μ g of two DNA oligonucleotide fragments is mixed, 100 ℃ of sex change 5 minutes, the renaturation of slowly lowering the temperature is then used T4 phage polynucleotide and is swashed acid and carry out phosphorylation after the renaturation.The pXC.1 carrier segments of connecting joint after this phosphorylation with disappearance 2809-3329bp zone is connected called after pXC-del E1b (method is referring to the molecular cloning experiment guide, and scientific publication 1992 is published).Synthetic primer in the position is respectively near inserting the connecting joint two ends
03 CTG?GCC?AAT?ACC?AAC?CTT?A
04 ATA?TGA?GCT?CAC?AAT?GCT?TC
PXC-del E1b is carried out polymerase chain reaction (PCR) carry out amplification in vitro (method is referring to the molecular cloning experiment guide; And scientific publication 1992 is published); Its PCR product checks order. result shows: CTGGCCAATACCAACCTTATCCTACACGGTGTAAGCTTAATGAGTAACTAAGATCT GGAAGGTGCTGAGGTACGATGAGACCCGCACCAGGTGCAGACCCTGCGAGTGTGGC GGTAAACTATTAGGAACCAGCCTGTGATGCTGGATGTGACCGAGGAGCTGAGGCCC GATCACTTGGTGCTGGCCTGCACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATAC AGATTGAGGTACTGAAATGTGTGGGCGTGGCTTAAGGGTGGGAAAGAATATATAAG GTGGGGGTCTTATGTAGTTTTGTATCTGTTTIGCAGCAGCCGCCGCCGCCATGAGC ACCAACTCGTTTGATGGAAGCATTGTGAGCTCATAT, this shows that pXC-delE1b has lacked the 2809-3329 zone and insert TAATGAGTAACTAA in this zone in the pXC.1 carrier.
Defective virus transfection of the present invention is transformed people embryo retina cell strain 911 cells of human embryonic kidney cell's strain 293 or E1 conversion to E1, example of the present invention is with its rotaring redyeing 293 cell, 293 cell strains are purchased in Canadian Microbix Biosystem Inc. (Toronto), be to transform the human embryonic kidney cell by the 5 type adenovirus DNAs of shearing to form, it contains and expresses 5 type adenovirus E 1 districts, and adenovirus DNA has high transfection efficiency to it.The plasmid that will contain 5 type adenovirus left arms is united plasmid co-transfection 293 cells that contain 5 type adenovirus right arms, can produce the adenovirus with infectivity by homologous recombination.We pass through the thin strain of Lipofectamine cotransfection to 293 with pXC-del E1b and plasmid pBHG10 that contains 5 type adenovirus right arms or pBHGE3, and its concrete grammar is referring to the operation instructions of GIBCO BRL company.PBHG10 and pBHGE3 purchase in Canadian Microbix Biosystems Inc. (Ontario), and pBHG10 contains 5 type adenovirus right arms, but disappearance E3 district; PBHGE3 contains 5 type adenovirus right arms, contains the E3 district.Virus plaque appearred behind the cotransfection in 9-14 days, through three virus plaque purifying, this adenovirus called after CNHK201 and CNHK202, concrete grammar is referring to GeneTransfer and Expression Protocols, Murray EJ chief editor, Humana Press 1991 publishes.
Example two. the generation and the genome structure of virus
The recombinant adenovirus method is referring to example one content.Its title is as follows:
Virus Name contains Ad5 left arm plasmid and contains Ad5 right arm plasmid
Ad5-del?Elb?E3 CNHK201 pXC-del?E1b PBHG10
Ad5-del?E1b CNHK202 pXC-del?E1b PBHGE3
Adenovirus is breeding in a large number in 293 cells, uses the method large-scale purification adenovirus (concrete grammar is referring to Gene Transfer and ExpressionProtocols, and Murray EJ edits, and Humana Press 1991 publishes) of caesium chloride density gradient centrifugation.Its insertion sequence confirms by order-checking.Ad5-del E1b E3 (CNHK201) is 5 type adenovirus, (E1b 55Kda protein gene partial sequence) deletion mutantion in the 2809-3329bp zone, and in the deletion mutantion zone, insert the sequence TAATGAGTAACTAA contain two terminator codons, with 28133-30818bp (E3 district partial sequence), other dna sequence dnas of virus are identical with 5 type adenovirus simultaneously.Ad5-del E1b (CNHK202) is 5 type adenovirus, (E1b 55Kda protein gene partial sequence) deletion mutantion in the 2809-3329bp zone, and in the deletion mutantion zone, inserting the sequence TAATGAGTAACTAA that contains two terminator codons, other dna sequence dnas of virus are identical with 5 type adenovirus.
Claims (4)
1. defective adenoviral, the E1b 55Kda protein gene excalation of adenovirus is also inserted terminator codon at the disappearance position, it is characterized by adenovirus 2809-3329bp disappearance, and dna fragmentation TAATGAGTAACTAA is inserted at this disappearance position.
2. defective adenoviral according to claim 1 is characterized in that this recombinant adenovirus can form mixture with one of following compounds:
(1). chemotherapeutic agent
(2). biotoxin.
(3). monoclonal antibody.
3. the construction process of a defective adenoviral makes up the back transfection to cell, it is characterized in that (1). E1b 55Kda protein gene 2809-3329bp excalation in two adenovirus carriers and disappearance district are inserted terminator codon TAATGAGTAACTAA; (2). with above-mentioned two adenovirus carrier transfections to reconstitution cell; (3). separate above-mentioned recombinant adenovirus.
4. the construction process of the described defective adenoviral of root a tree name claim 3 is characterized in that people embryo retina cell strain 911 cells of its reorganization back Adenovirus Transfection to E1 conversion human embryonic kidney cell's strain 293 or E1 conversion.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99124030A CN1254719A (en) | 1999-11-19 | 1999-11-19 | Defective adenovirus and its building-up method |
| PCT/CN2000/000428 WO2001037867A1 (en) | 1999-11-19 | 2000-11-17 | A new defective adenovirus and process for producing said adenovirus |
| AU13794/01A AU1379401A (en) | 1999-11-19 | 2000-11-17 | A new defective adenovirus and process for producing said adenovirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99124030A CN1254719A (en) | 1999-11-19 | 1999-11-19 | Defective adenovirus and its building-up method |
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| Publication Number | Publication Date |
|---|---|
| CN1254719A true CN1254719A (en) | 2000-05-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99124030A Pending CN1254719A (en) | 1999-11-19 | 1999-11-19 | Defective adenovirus and its building-up method |
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|---|---|
| CN (1) | CN1254719A (en) |
| AU (1) | AU1379401A (en) |
| WO (1) | WO2001037867A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002095041A1 (en) * | 2001-05-25 | 2002-11-28 | Qijun Qian | Virus high effectively expressing the tumor interferon and specifically reproduction in tumor cells and the use thereof |
| CN1880457B (en) * | 2001-10-11 | 2010-05-26 | 麦克公司 | Ad6 recombinant nucleic acid |
| CN101781636A (en) * | 2009-01-19 | 2010-07-21 | 中国人民解放军第二军医大学东方肝胆外科医院 | Proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene, construction method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2705686B1 (en) * | 1993-05-28 | 1995-08-18 | Transgene Sa | New defective adenoviruses and corresponding complementation lines. |
| FR2725726B1 (en) * | 1994-10-17 | 1997-01-03 | Centre Nat Rech Scient | VIRAL VECTORS AND USE IN GENE THERAPY |
-
1999
- 1999-11-19 CN CN99124030A patent/CN1254719A/en active Pending
-
2000
- 2000-11-17 WO PCT/CN2000/000428 patent/WO2001037867A1/en active Application Filing
- 2000-11-17 AU AU13794/01A patent/AU1379401A/en not_active Abandoned
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002095041A1 (en) * | 2001-05-25 | 2002-11-28 | Qijun Qian | Virus high effectively expressing the tumor interferon and specifically reproduction in tumor cells and the use thereof |
| CN1880457B (en) * | 2001-10-11 | 2010-05-26 | 麦克公司 | Ad6 recombinant nucleic acid |
| CN101781636A (en) * | 2009-01-19 | 2010-07-21 | 中国人民解放军第二军医大学东方肝胆外科医院 | Proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene, construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001037867A1 (en) | 2001-05-31 |
| AU1379401A (en) | 2001-06-04 |
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