CN101082042A - Application of artificial reconstructed defect type human 3-type adenovirus - Google Patents
Application of artificial reconstructed defect type human 3-type adenovirus Download PDFInfo
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- CN101082042A CN101082042A CN 200610035667 CN200610035667A CN101082042A CN 101082042 A CN101082042 A CN 101082042A CN 200610035667 CN200610035667 CN 200610035667 CN 200610035667 A CN200610035667 A CN 200610035667A CN 101082042 A CN101082042 A CN 101082042A
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Abstract
The present invention discloses one kind of artificially reconstructed defected human type-III adenovirus capable of being used as vaccine or gene treating carrier. The defected human type-III adenovirus containing intact virus shell and defected genome may be copied in special packing cell and may be not proliferated in host cell. It may be applied in immunizing human body to generate specific protecting antibody against type-III adenovirus and prevent infection of type-III adenovirus. Being incapable of being copied in human body, the defected human type-III adenovirus has no pathogenicity and high safety. The defected human type-III adenovirus may be used as carrier for inserting exogenous gene, proliferated in special packing cell to express exogenous protein and used for gene therapy.
Description
Technical field
The invention belongs to the Application Areas of engineered virus aspect vaccine, tumour and gene therapy.Adenovirus by expression alien gene, having very wide application prospect aspect oncotherapy and the gene therapy, can be widely used in corresponding fields such as medical science, biology as expression vector; Develop the adenovirus vaccine of highly effective and safe simultaneously, significant aspect prevention adenovirus related breathing tract disease.
Background technology
The adenovirus of finding has 51 serotypes at present, and it is that a class mainly infects human airway and gastral pathogenic agent.Human adenovirus can cause inflammation and tonsilla, gland tissue and the adenoid recessiveness or the persistent infection of multiple tissues such as acute pharyngitis, infant's lethality pneumonia, epidemic conjunctivitis, urocystitis, enteritis.Adenovirus infection can be widely current in the world, infectivity is strong.Adenovirus infection is also very general in China, and adenovirus pneumonia is the highest a kind of virus pneumonia of China's sickness rate.
In treatment, adenovirus infection does not equally have specific medicament with other virus diseases, generally all can only take symptomatic treatment, mainly is to fully recover by the immune opposing reaction of body self, elimination virus.China does not still prevent the development of the vaccine of adenovirus infection at present, is still belonging to blank aspect this.Studies show that the adenovirus antibody that body produces has permanent and stable protectiveness to homologous virus.Therefore, if can stimulate body to produce the antibody of certain level, then can play the effect of this hypotype adenovirus infection of prevention with adenovirus coat protein.
On the other hand, adenovirus is most widely used a kind of virus vector in the present gene therapy, and it has following advantage: host cell is in extensive range, not only can infect somatoblast but also can infect Unseparated Cell; Preparation infectious titer particle can reach 10 easily
11Pfu/ml; And in being subjected to transfect cell, do not integrate, therefore there is not carcinogenic and mutagenic danger; Can insert big segmental foreign gene.The adenovirus carrier of developing at present mainly is people's 5 type adenovirus.Yet 5 type adenovirus infection cells need specific acceptor (COxsackie-adenovirus receptor) to exist, and some cells lack corresponding acceptor, and efficiency of infection is very low.And that the acceptor of 3 type adenovirus (CD46) exists is wider, is that carrier can infect various kinds of cell with 3 type adenovirus, uses wider.
The above condition provides solid basis for engineered defect type human 3-type adenovirus in the application prospect aspect gene therapy vector and the vaccine development.
Summary of the invention
The object of the present invention is to provide a kind of defect type human 3-type adenovirus, it can be used as vaccine or adenovirus carrier.
In order to solve above-mentioned task, the present invention takes following measure:
(1) breeds human 3-type adenovirus clinical separation strain (Guangzhou Children's Hospital's preservation) in a large number, extract virus genom DNA, carry out the special genes operation, make viral E1 genetically deficient, make it to lose ability in normal host cell internal breeding.This defective gene group DNA transfection is to providing accordingly in the packing cell of missing gene protein expression, carry out the packing of adenovirus particles, the virus that is packaged to be possesses complete virus coat, but genome is an excalation, can not breed in normal host cell.
(2) defective adenoviral of purifying and being packaged to be carries out human immunity as vaccine, can prevent the infection of human 3-type adenovirus.
(3) this defect type human 3-type adenovirus also can be used as carrier, inserts foreign gene, expression alien gene or as gene therapy in specific packing cell.
The present invention compared with prior art has following advantage:
The adenovirus of the replication defect type that we develop; though because of its genomic defective reproducible not; but the shell that in corresponding packing cell, still has complete wild-type adenovirus; so still have the somatic ability of infected person; can be by infecting body; bring out human body and produce the purpose that reaches immunoprophylaxis at the specificity protection antibody of human 3-type adenovirus, but cause disease because of it no longer duplicates can not breed, higher than common attenuated vaccine security.Simultaneously can expression alien gene in specific packing cell.
Embodiment
Embodiment 1: the structure of defect type human 3-type adenovirus
(1) cultivate the HEp-2 cell, the propagation human 3-type adenovirus extracts the full genome of virus.
(2) pcr amplification 3 type adenovirus 1-580bp (AdL1) and 3000-6354bp (AdL2) fragment.
(3) connecting AdL1 and AdL2 is AdL.
(4) the SalI enzyme is cut the adenoviral gene group, reclaims the big fragment of 6354bp-35273bp (AdR).
(5) connect AdL and AdR, obtain AdLR, transfection is in the specific HA cell that contains 3 type adenovirus E 1 district gene fragments.
(6) in the HA cell, carry out homologous recombination, utilize the plaque isolation technique to separate 3 type adenovirus of the defective type of reorganization.
(7) utilize fluorescent quantitative PCR technique to determine to separate whether the virus that obtains is defective type.
(8) inoculation separates the virus obtain to the HEp-2 cell, determines whether virus can self-replacation.
(9) breed recombination deficient mutant 3 type adenovirus in a large number, work done in the manner of a certain author is vaccine behind the viral purification.
Embodiment 2: the defect type human 3-type adenovirus expression of exogenous gene
(1) pcr amplification gene order to be expressed is inserted eukaryotic expression cassette, contains CMV promotor and PolyA.
(2) two ends connect AdL1 and AdL2 fragment.
(3) advance to contain in the HA cell of 3 type adenovirus E 1 district gene fragments with defective type 3 type adenoviral gene group AdLR cotransfections, carry out homologous recombination.
(4) fluorescent quantitative PCR technique screening contains and remains the recombination deficient mutant adenovirus of expressing gene sequence.
(5) breed this adenovirus in a large number, use as gene therapy behind the viral purification; Perhaps direct purification goes out this foreign gene of expressing in the HA cell.
Claims (2)
1, a kind of defect type human 3-type adenovirus is characterized in that:
(a) viral E1 Gene Partial disappearance;
(b) this defective virus can't be bred in normal host cell, can only duplicate in the specific packing cell that the missing gene protein expression is provided;
(c) in packing cell, can produce complete virus particle;
(d) its genome of the adenovirion that produces in packing cell is incomplete, but still possesses infectivity.
(e) this virus can infect human body, causes immune response, stimulates human body to produce specific antibody, but can not breed in human body cell.
2, a kind of defect type human 3-type adenovirus according to claim 1 is characterized in that:
This defect type human 3-type adenovirus can be used as vaccine or carrier, inserts foreign gene, expression alien gene or as gene therapy in specific packing cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610035667 CN101082042A (en) | 2006-05-29 | 2006-05-29 | Application of artificial reconstructed defect type human 3-type adenovirus |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200610035667 CN101082042A (en) | 2006-05-29 | 2006-05-29 | Application of artificial reconstructed defect type human 3-type adenovirus |
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| CN101082042A true CN101082042A (en) | 2007-12-05 |
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| CN 200610035667 Pending CN101082042A (en) | 2006-05-29 | 2006-05-29 | Application of artificial reconstructed defect type human 3-type adenovirus |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619324A (en) * | 2008-07-01 | 2010-01-06 | 中国疾病预防控制中心病毒病预防控制所 | Replication defective recombinant adenovirus Ad41 vector system and application thereof |
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2006
- 2006-05-29 CN CN 200610035667 patent/CN101082042A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619324A (en) * | 2008-07-01 | 2010-01-06 | 中国疾病预防控制中心病毒病预防控制所 | Replication defective recombinant adenovirus Ad41 vector system and application thereof |
| CN101619324B (en) * | 2008-07-01 | 2013-06-26 | 中国疾病预防控制中心病毒病预防控制所 | Replication defective recombinant adenovirus Ad41 vector system and application thereof |
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