CN1704477A - Alpha (1,3) galactose transferase recombinant cattier and its production and use - Google Patents
Alpha (1,3) galactose transferase recombinant cattier and its production and use Download PDFInfo
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- CN1704477A CN1704477A CN 200410042649 CN200410042649A CN1704477A CN 1704477 A CN1704477 A CN 1704477A CN 200410042649 CN200410042649 CN 200410042649 CN 200410042649 A CN200410042649 A CN 200410042649A CN 1704477 A CN1704477 A CN 1704477A
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Abstract
The invention relates to an alpha (1,3) galactose transferase expression vector, which can effectively infect various human tumor cells, make them express alpha (1,3) galactosyltransferase, thus resulting the tumor cell surface expression of alpha-Gal galactose epitope, and increasing the exposure of the tumor specific antigen epitope, the immunologic rejection reaction can be initiated, and tumors can be prevented and/or treated. The preparing process mainly comprises gene cloning, expression, purifying and restructuring.
Description
Technical field the present invention relates to biomedical sector, specifically relate to a kind of α (1,3) galactotransferase expression vector, feature is efficient various human tumor cells, makes it express alpha (1,3) galactotransferase, cause tumor cell surface expression α-Gal semi-lactosi epi-position, increase the exposure of tumour specific antigen epi-position simultaneously, cause immunological rejection, prevention is or/and true tumors such as treatment tumours.The main applying gene clone of preparation method expresses purifying, genetic and cell engineering technology such as reorganization.This carrier can be used for the gene therapy and the immunotherapy of human multiple disease, can effectively treat or/and prevent true tumors such as human kinds of tumors.
Background technology is along with the progress of Protocols in Molecular Biology, and the particularly development of genetic and cell engineering technology, neoplastic gene therapy such as tumour become the focus of people's research.At present, the whole world has 600 multinomial gene therapy schemes and gets permission to enter clinical experiment, and some treatment plan has obtained certain curative effect, has tentatively shown the prospect of gene therapy.As the various carriers that can be used for gene therapy, itself does not have the meaning of any treatment disease, no clinical value; And as can be used for the goal gene of various treatments,, only have the potential therapeutic action owing to directly import target cell and acquiring a certain degree of difficulty of in target cell, directly expressing thereof, there is not actual clinical value.Have only the gene that will have potential therapeutic action to combine,, goal gene is imported target cell and expression, just can reach real clinical therapeutic efficacy by latter's mediation with the carrier of transferable gene.Therefore, the fusion sequence of structure therapeutic gene and genophore is the key that realizes gene therapy.It is to carry out homologous recombination in eukaryotic cell that the expression cassette of goal gene and carrier are merged method commonly used, the process complexity, waste time and energy, use the genetic engineering technique of in prokaryotic cell prokaryocyte (intestinal bacteria), realizing homologous recombination, vector construction hexose transport protein then can effectively address the above problem.
Lack specificity, the gene transfer vector of target and high efficiency is a difficult problem of therapy of tumor always, at present, is used for Vectors in Gene Therapy and mainly is divided into virus vector and non-virus carrier two big classes.Virus vector commonly used has adenovirus carrier, gland relevant viral vector and retroviral vector etc.Adenovirus carrier is the genophore of using always, its advantage is that transfection efficiency height, operability are good, can carry bigger goal gene segment, can prepare the virion that height is tired, be easy to suitability for industrialized production, can infect the division stage cell, also can infect non-division stage cell, have safety, pathogenic advantage such as low.Weak point is also arranged, the one, infect the shortage specificity, the 2nd, have immunogenicity.Therefore, to adenovirus carrier deposit profit go the improvement of fraud be the development gene therapy the only way.Studies show that, carry allogenic gene and be integrated into the expression that realizes the long period of goal gene in the target cell genome in adenovirus carrier E1 or E3 disappearance district, and can reduce its immunogenicity, retroviral vector can carry exogenous origin gene integrator and advance in the target cell genome to realize that goal gene stablizes persistent expression, but the external breeding titre of retrovirus is low, transfection efficiency is low, only infecting the division stage cell, is random integration in karyomit(e); All there are different pros and cons in other virus vector that can be used for transgenosis with non-virus carrier.
A large amount of evidence prompting human body internal memories are at preformed antibody, and promptly natural antibody (NAb) is the basis that there be hyperacute rejection phenomenon (HAR) in heteroplastic graft.The natural antibody of people among the HAR can extensively discern pig antigen, but in fact, complement-dependent rejection natural antibody in the 90% above human serum only discerns the antigenic structure of xenogenesis (pig), be α (1,3) galactosyl (gal), it is the antigenic epitope of xenogenesis (pig) (determinant), and these antigens all carry a α (1,3) galactosyl epitope specifically.The formation of α (1,3) galactosyl, mainly by α (1,3) galactotransferase catalysis, this kind of enzyme is at lower animal and New World monkey expression in vivo, people, ape, and old world monkey body in all lack this kind of enzyme.Because human body express alpha (1,3) galactotransferase not, do not carry the epitope (determinant) of α (1,3) semi-lactosi yet, therefore contact with this antigen continually and make intravital natural antibody that very high tiring be arranged.People's cell, tumour cell particularly, introduce the α (1 of xenogenesis (pig), 3) galactotransferase gene makes it express alpha (1,3) galactotransferase, cause tumor cell surface great expression semi-lactosi epi-position, the semi-lactosi epi-position has caused the cytotoxicity of the intravital natural antibody of people to tumour cell generation complement-mediated, and then the exposure of tumour specific antigen initiation specific immune response, reaches treatment or/and the purpose of prophylaxis of tumours.This method makes full use of the natural antibody that exists in the body, is a kind of easy, and economic gene therapy is or/and the immunotherapy scheme.
Summary of the invention the present invention is intended to organically combine having the gene of potential therapeutic action and the carrier of transferable gene, and provide a kind of virus vector and α (1,3) recombinant vectors of galactotransferase gene, can in the specific cells of genetic engineering modified mistake, breed, produce, and can in eukaryotic cell, express, make the tumor cell membrane surface produce more α (1,3) galactosyl epi-position, be used for the gene therapy and the immunotherapy of human multiple disease.The result is encouraging, cell infection α (1,3) after the recombinant virions of galactotransferase gene, cell surface produces than the galactosan epi-position, caused the intravital natural antibody of people tumour cell has been produced the specific immune response that the exposure of the cytotoxicity of complement-mediated and tumour specific antigen causes, reached treatment or/and the purpose of prophylaxis of tumours.The present invention also aims to provide the preparation method of recombinant vectors and be used to prepare treatment or/and the method for prophylaxis of tumours medicine.
For achieving the above object, the invention provides a kind of virus vector and α (1,3) recombinant vectors of galactotransferase gene, this recombinant vectors is with virus vector and α (1 by the dna clone technique construction, 3) the galactotransferase expression casette combines, be built into an energy and in the specific cells of genetic engineering modified mistake, increase, breed, also can be in eukaryotic cell the fusion sequence of express alpha (1,3) galactotransferase.
The carrier of this recombinant vectors can be plasmid vector, dna virus or rna virus vector any, and its preferred vector is adenovirus carrier or the complex carrier that contains the adenovirus carrier sequence; Most preferably carrier is an adenovirus carrier.
Described α (1,3) galactotransferase gene can be any of α (1,3) galactotransferase gene, and most preferred gene is pig α (1, a 3) galactotransferase gene.
This invention provides the methods for making and using same of this recombinant vectors:
One, recombinant alpha (1,3) galactotransferase gene recombined vector and/or recombinant adenovirus particulate preparation method provide a kind of with gene clone in the molecular biology, express, purifying, genetic and cell engineering technology such as reorganization prepare the method for recombinant vectors.The carrier of this recombinant vectors can be any of dna virus or RNA viruses, and its preferred vector is adenovirus carrier or the complex carrier that contains the adenovirus carrier sequence; Most preferably carrier is an adenovirus carrier.This method is introduced α (1,3) galactotransferase gene in carrier, form transfection efficiency height, workable recombinant alpha (1,3) galactotransferase gene recombinant adenovirus vector and/or recombinant adenovirus particle.
Two, application method the present invention of recombinant vectors or virosome provides and has used above-mentioned recombinant virions and carry out the treatment of true tumor such as tumour or/and the method for prevention.Recombinant vectors of the present invention or virosome can be used for being prepared into suitable formulation, as liquid dosage form, semisolid dosage form, solid type or gas formulation.The promotor of therapeutic gene can replace with the human organ specificity promoter in the recombinant vectors of the present invention, reaches the purpose of magnetic target therapy.Recombinant vectors of the present invention can be used for tumour cell is carried out immune modification, makes the tumor vaccine prophylaxis of tumours.
The present invention will be further described below in conjunction with embodiment, all this areas of having done according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Embodiment
Embodiment 1
For example, the recombinant alpha of pig (1,3) galactotransferase gene adenovirus body (particle) is prepared like this:
(a) in adenovirus carrier (as pShuttle) sequence, fixed point is introduced the cDNA of α (1, the 3) galactotransferase of pig, forms α (1,3) galactotransferase gene adenovirus shuttle vector;
(b) change α (1,3) galactotransferase gene adenovirus shuttle vector in (a) in the prokaryotic cell prokaryocyte (as BJ5183-AD-1) screening, picking positive colony, enzyme are cut and are checked order and determine correct positive colony, and increase and obtain recombinant vectors DNA;
(c) the recombinant vectors DNA with amplification in (b) changes packing cell (as Ad-293) over to, the adenovirus body (virion of replication defective) of the α of an amount of pig of output (1,3) galactotransferase gene;
(d) above-mentioned virion purifying is prepared into preparation, as liquid dosage form, semisolid dosage form, solid type or gas formulation.
Embodiment 2
For example, the recombinant alpha of pig (1,3) galactotransferase gene adenovirus body treatment pointed condyloma is implemented like this:
(a) recombinant alpha of pig (1,3) galactotransferase gene adenovirus body is prepared into the semi-solid preparation that quality meets the clinical trial standard; Then
(b) semi-solid preparation in the pointed condyloma surface applied (a) causes pointed condyloma cell surface great expression α (1,3) galactosyl epi-position, excites immunological rejection, reaches the purpose of removing and/or preventing pointed condyloma.
Embodiment 3
For example, the recombinant alpha of pig (1,3) galactotransferase gene adenovirus body intratumor injection is implemented like this:
(a) recombinant alpha of pig (1,3) galactotransferase gene adenovirus body is prepared into the injection liquid that quality meets the clinical trial standard; Then
(b) the middle injection liquid of direct injection (a) in the superficial tumor knurl body, in-vivo tumour is injected injection liquid in (a) with the interventional method west in tumour knurl body, cause tumor cell surface great expression α (1,3) galactosyl epi-position, excite immunological rejection, reach the purpose of removing and/or prophylaxis of tumours.
Embodiment 4
The transformation of the recombinant alpha of pig (1,3) galactotransferase gene adenovirus body prostate gland target is implemented like this:
(a) promotor of clone purification people's prostate specific antigen gene, the regulation and control test confirms effectively; Then
(b) correct directed cloning is gone into recombinant alpha (1,3) the galactotransferase gene adenovirus recombination vector of pig, substitutes original promotor, finishes recombinant alpha (1,3) the galactotransferase gene adenovirus body prostate gland target transformation of pig.
Embodiment 5
The recombinant alpha of pig (1,3) galactotransferase gene adenovirus body is modified tumour-cell vaccine and is prepared like this:
(a) cultivate the amplification tumour cell, modify with recombinant alpha (1,3) the galactotransferase gene adenovirus body transfection of pig;
(b) screening transfection tumor cell's positive colony or do not screen;
(c) physics or chemical process are advanced inhibition effective period;
(d) making the tumour-cell vaccine preparation uses.
Claims (10)
1. recombinant vectors, be vector gene and α (1,3) galactotransferase is expressed the recombinant chou of coding region, prepare by the genetic and cell engineering method, can be in target cell or host cell express alpha (1,3) galactotransferase is used for the magnetic target therapy of guidances such as non-specific or organ specific promoters and is used for tumour cell is carried out immune modification, makes the tumor vaccine prophylaxis of tumours.
2. the described carrier of claim 1, it is characterized in that: the carrier of this recombinant chou can be plasmid vector, dna virus or rna virus vector any, its preferred vector is adenovirus carrier or the complex carrier that contains the adenovirus carrier sequence; Most preferably carrier is an adenovirus carrier.
3. the described carrier of claim 2, it is characterized in that: comprise α (1,3) galactotransferase gene expression frame, the early stage polyadenylic acid signal of cytomegalovirus IE promotor and SV40, α (1,3) galactotransferase genetic expression is controlled by non-specific promotors such as cytomegalovirus IE or the organ specific promoters such as promotor of prostate specific antigen are controlled.
4. the described carrier of claim 2, it is characterized in that: the necessary genetically deficient of adenoviral replication, introduce α (1,3) galactotransferase gene expression frame, described α (1,3) galactotransferase gene can be any of α (1,3) galactotransferase gene, most preferred gene is pig α (1, a 3) galactotransferase gene.
5. the described carrier of claim 2 is characterized in that: the E1A of adenovirus carrier and E1B district disappearance, introducing α (1,3) galactotransferase gene expression frame.
6. the described carrier of claim 1 is characterized in that: adopt any suitable acceptable pharmaceutical formulation or/and administering mode during application.
7. save wild-type α (1,3) method of the cell of galactotransferase afunction is: the described recombinant vectors of a certain amount of claim 1 is by contact and enter wild-type α (1,3) cell of galactotransferase genetically deficient, effective expression wild-type α (1,3) galactotransferase albumen is saved cell in cell.
8. the described method of claim 7, it is characterized in that: the cell of α (1,3) galactotransferase afunction is the cell of α (1,3) galactotransferase genetically deficient, the cell of α (1,3) galactotransferase afunction is people or mammalian cell.
9. method of producing recombinant adenovirus comprises: the adenoviral plasmid carrier that (a) will contain express alpha (1,3) galactotransferase element imports proper host cell with liposome-mediated transfection method; And (b) analyze the cytopathic effect of the host cell appearance of cultivating, the viral yield of monitoring homologous recombination.
10. the described method of claim 9 is characterized in that: this adenoviral plasmid has to duplicate and lacks limit, and host cell can remedy this defective; This adenoviral plasmid lacks functional E1A and E1B gene, and host cell is a functional performance E1 genetic expression cell; This host cell is 293 cells, and the substratum of this host cell is the MEM substratum.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016101326A1 (en) * | 2014-12-22 | 2016-06-30 | 赵永祥 | Oncolytic heterologous recombination newcastle disease virus,preparation method and application thereof |
| CN105821030A (en) * | 2015-01-04 | 2016-08-03 | 彭霞 | Cancer cell/dendritic cell fusion tumor vaccine for expression of alpha1,3 galactosyl transferase and preparation method thereof |
| CN105821029A (en) * | 2015-01-04 | 2016-08-03 | 彭霞 | Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof |
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2004
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016101326A1 (en) * | 2014-12-22 | 2016-06-30 | 赵永祥 | Oncolytic heterologous recombination newcastle disease virus,preparation method and application thereof |
| US10080797B2 (en) * | 2014-12-22 | 2018-09-25 | Yongxiang Zhao | Oncolytic heterologous recombinant newcastle disease virus, preparation method and application thereof |
| CN105821030A (en) * | 2015-01-04 | 2016-08-03 | 彭霞 | Cancer cell/dendritic cell fusion tumor vaccine for expression of alpha1,3 galactosyl transferase and preparation method thereof |
| CN105821029A (en) * | 2015-01-04 | 2016-08-03 | 彭霞 | Heterologous fusion gene modified cancer cell/dendritic cell fusion tumor vaccine and preparation method thereof |
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