CN103255170A - Oncolytic adenovirus construction method for targeted CD47 positive leukemia cells and application thereof - Google Patents
Oncolytic adenovirus construction method for targeted CD47 positive leukemia cells and application thereof Download PDFInfo
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Abstract
The invention discloses an oncolytic adenovirus construction method for targeted CD47 positive leukemia cells and application thereof. The method comprises the following steps of: constructing an expression frame for Scar-4N1; inserting the expression frame into a corresponding cleavage site of a pXC2-sp-E1A plasmid to obtain a pXC2-sCAR-4N1-sp-E1A plasmid; removing E1B from a pXC2 plasmid, and inserting an exogenous antioncogene expression frame into the pXC2 plasmid to obtain a Pxc2-deltaE1B-gene plasmid; and then constructing a pXC2-sCAR-4N1-sp-E1A-deltaE1B-gene plasmid. An oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or an oncolytic adenovirus Ad. sCAR-4N1-sp-E1A-deltaE1B-gene are used for preparing anticancer medicines. Experiments in vivo and in vitro show that the oncolytic adenovirus has a better effect of killing the leukemia cells CD47.
Description
Technical field
The present invention relates to the oncolytic adenovirus field of gene, especially relate to oncolytic adenovirus construction process of a kind of target CD47 Positive Leukemic Cells and uses thereof.
Background technology
Leukemia is the different substantiality disease that is caused by the growth of a series of and cell, numerous genovariations that differentiation is relevant with death, it is malignant tumor of hematopoiesis system, its sickness rate is 4.17/10 ten thousand in China, in tumour, occupy the 7th, American-European countries is 6.14/10 ten thousand, occupy the 6th of tumour, mortality ratio occupies the 6th of tumour, is one of malignant disease of serious harm human body health.The development of chemotherapy technology has improved total lifetime of leukaemic greatly, but recurrence remains a great problem that leukemia treating faces, especially gerontal patient and has the cytogenetic patient of poorer prognosis.In recent years, existing increasing clinical and experimental study demonstration, exist the few leukemic stem cells of a group ratio (Leukemia stem cells in leukaemic's body, LSCs) decisive role in starting leukemia generation and development, the main root that is considered to drug-resistant leukemia and recurrence has only the leukemic stem cells of removing just might effect a radical cure leukemia.
Leukemic stem cells proportion in the leukemia cell is extremely low, but has certain self and differentiation potential, in leukemic generation and development vital role is arranged.Based on the targeted therapy of leukemic stem cells, eliminate leukemic stem cells, eradicate the possibility of leukemia relapse from the source, be expected to become the leukemic New Policy of healing.
Very ripe to the research of 5 type adenovirus (Ad5) at present, so Ad5 becomes the main adenovirus carrier for therapy of tumor.COxsackie-adenovirus receptor (Coxsakie and adenovirus receptor on the infection height dependent cells surface of the cell of Ad5, CAR), but the tumor cell surface of the epithelial cell source property of leukemia cell and nearly 50% lacks CAR, makes Ad5 be difficult to infect these cells.And on the other hand, tumour cell again often high expression level some be different from most Normocellular membranins, as acute myeloid leukaemia (Acute myeloid leukemia, and most of normal hematopoiesis stem cell is not expressed these albumen AML) and stem cell (AML LSC) high expression level CD123, CD44 and CD47.Before this, we utilize CD123 to design the oncolytic adenovirus targeted drug as the leukemia treating target spot, this oncolytic adenovirus is by leukemia cell's surface C D123 cells infected, massive duplication in cell, great expression antioncogene and then lysing cell reach the purpose (Chinese patent publication number CN 102747046 A) of targeted therapy with this.Yet leukemia is a kind of heterogeneous hematologic cancers, and AML LSC is a kind of heterogeneous cell colony, and only the therapeutic modality at a kind of target spot obviously is difficult to satisfied actual needs clinically, but lacks the method for combination therapy at present.
Summary of the invention
One of purpose of the present invention is to set up a hull fibres (Fiber) by modification Ad5 oncolytic adenovirus makes it enter the system of leukemia cell, lysing cell via CD47.This system is based on the principle of part (Ligand) and acceptor (Receptor) specific recognition, make the Ad5 oncolytic adenovirus carry the sCAR-4N1 fusion gene, sCAR(soluble CAR) be the film outer segment of CAR, 4N1 is that thrombospondin TSP-1 goes up and the interactional zone of CD47.Oncolytic adenovirus is namely expressed this fusion rotein in the assembling process in packing cell, combination by sCAR and Fiber is assembled into the oncolytic adenovirus shell, 4N1 part then can specific recognition CD47, therefore the leukemia cell that the oncolytic adenovirus that obtains can the special infection CD47 positive.Oncolytic adenovirus not only can copy in target cell after infecting the CD47 Positive Leukemic Cells, can also continue to express the sCAR-4N1 fusion rotein, finish the modification of oncolytic adenovirus shell, therefore duplicated new virus can start the new round infection to target cell, to reach high infection rate.This oncolytic adenovirus can use separately according to concrete clinical case, perhaps unites use with the oncolytic adenovirus of target CD123, to reach desirable curative effect.
The construction process of the oncolytic adenovirus of target CD47 Positive Leukemic Cells, the oncolytic adenovirus that described method makes up carries the sCAR-4N1 expression cassette.
The oncolytic adenovirus portability external source antioncogene expression cassette that described method makes up.
The construction process of the oncolytic adenovirus of described target CD47 Positive Leukemic Cells, it may further comprise the steps:
1) expression cassette of structure sCAR-4N1;
2) enzyme cuts back to close the expression cassette of sCAR-4N1, is inserted into the corresponding restriction enzyme site of pXC2-sp-E1A plasmid, obtains the pXC2-sCAR-4N1-sp-E1A plasmid;
3) the pXC2 plasmid is introduced restriction enzyme site in situ behind deletion E1B, inserts external source antioncogene expression cassette structure and obtains pXC2-△ E1B-gene plasmid; Cut connection by enzyme the sCAR-4N1-sp fragment on the pXC2-sCAR-4N1-sp-E1A plasmid is replaced on the pXC2-△ E1B-gene plasmid, make up and obtain pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid;
4) pXC2-sCAR-4N1-sp-E1A plasmid and pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid respectively with pBHGE3 skeleton plasmid cotransfection packing cell, in packing cell, finish the assembling of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene;
5) separation and purification oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene.
Described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are the condition replication type adenovirus that is copied by the control of tomour specific promotor.
Described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are the condition replication type adenovirus that obtains by the deletion virogene.
Described external source antioncogene comprises one or more among IL-24, TRAIL, MnSOD, smac, p53, the PPA.
Described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are for the preparation of antitumor drug.
Described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene and oncolytic adenovirus Ad-sCAR-IL3-sp-E1A and/or oncolytic adenovirus Ad-sCAR-IL3-sp-E1A-△ E1B-gene combined preparation antitumor drug.
The present invention has following beneficial effect:
(1) specific recognition according to part and acceptor designs, and the leukemia cell who expresses CD47 is had targeting preferably.
(2) the sCAR-4N1 fusion gene is loaded in the genome of oncolytic adenovirus, and this fusion gene can just be expressed when the packing cell inner virus is packed, and modifies the oncolytic adenovirus surface in non-covalent mode.
(3) the sCAR-4N1 fusion gene is owing to be the genome that is loaded in oncolytic adenovirus, therefore when oncolytic adenovirus infected tumor cell and when time multiplexed cell is processed, this fusion gene can obtain expresses and modifies the oncolytic adenovirus surface, therefore the surface of the new virus that copies out all will be assembled with the sCAR-4N1 fusion rotein, can further infect more CD47+ leukemia cell, reach high infection rate.
(4) oncolytic adenovirus of Gou Jianing has the site of inserting for antioncogene, and antioncogene is realized the efficient kill capability to tumour cell along with its copy number of virus replication raises significantly.
(5) according to the difference of antioncogene, can form a series of Ad.sCAR-4N1-sp-E1A-△ E1B-gene, as arms IL-24 antioncogene, then form Ad.sCAR-4N1-sp-E1A-△ E1B-IL24.
(6) working conditions rf oncolytic adenovirus, therefore the oncolytic adenovirus that makes up can copy at the tumour cell inner height, but can not copy in normal cell, realizes normal cell nontoxicity or hypotoxicity are reached the purpose of neoplasm targeted therapy with this.
(7) dissimilar condition rf oncolytic adenovirus carriers can be further used, the oncolytic adenovirus of a series of sCAR-4N1 of carrying and antioncogene expression cassette can be formed, with the leukemia cell of the target CD47 positive.
(8) can with the oncolytic adenovirus medicine of other target, unite use as the oncolytic adenovirus of target CD123.
Certainly implement arbitrary technical scheme of the present invention and might not reach above-described all beneficial effects simultaneously.
(9) but described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene and oncolytic adenovirus Ad-sCAR-IL3-sp-E1A and/or oncolytic adenovirus Ad-sCAR-IL3-sp-E1A-△ E1B-gene combined preparation antitumor drug.
Description of drawings
Fig. 1 is the construction process schematic flow sheet of pXC2-sCAR-4N1-sp-E1A plasmid and pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid.Wherein pXC2-sCAR-4N1-sp-E1A makes up the XhoI site that the sCAR-4N1 expression cassette inserts pXC2-sp-E1A to form; PXC2-sCAR-4N1-sp-E1A-△ E1B-gene carries out reclaiming the fragment that contains sCAR-4N1-sp behind NheI and the AleI double digestion to the pXC2-sCAR-4N1-sp-E1A plasmid, pXC2-△ E1B-gene is carried out same double digestion reclaim big fragment, the fragment of two recovery is coupled together to make up form.
Fig. 2 is the genome structure synoptic diagram of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and Ad.sCAR-4N1-sp-E1A-△ E1B-IL24.
Fig. 3 is that oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and the K562 leukemia cell's of Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 in-vitro multiplication suppresses capability analysis figure.
Fig. 3 A is that mtt assay detects the cell survival rate analysis chart, and data are represented with respect to the percentage of PBS group with dose gradient.
Fig. 3 B is that mtt assay detects the cell survival rate analysis chart, and data are represented with respect to the percentage of PBS group with time gradient.
Mtt assay detects and shows, oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 significantly suppress the in-vitro multiplication of leukemia cell K562 than Ad.sp-E1A, and Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 significantly is better than Ad.sCAR-4N1-sp-E1A, and its effect is dosage and time-dependent.The abbreviation of oncolytic adenovirus title, Ad.4N1:Ad.sCAR-4N1-sp-E1A; Ad.4N1-dBIL24:Ad.sCAR-4N1-sp-E1A-△ E1B-IL24.
Fig. 4 is that oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and the HL-60 leukemia cell's of Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 in-vitro multiplication suppresses capability analysis figure.
Fig. 4 A is that mtt assay detects the cell survival rate analysis chart, and data are represented with respect to the percentage of PBS group with dose gradient.
Fig. 4 B is that mtt assay detects the cell survival rate analysis chart, and data are represented with respect to the percentage of PBS group with time gradient.
Mtt assay detects and shows, oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 significantly suppress the in-vitro multiplication of leukemia cell HL-60 than Ad.sp-E1A, and Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 significantly is better than Ad.sCAR-4N1-sp-E1A, and its effect is dosage and time-dependent.The oncolytic adenovirus title same Fig. 3 that abridges.
Fig. 5. the inhibition ability of the K562 leukemia cell's transplanted tumor in nude mice of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-IL24.The oncolytic adenovirus title same Fig. 3 that abridges.
Embodiment
The construction process of the oncolytic adenovirus of target CD47 Positive Leukemic Cells, the oncolytic adenovirus that described method makes up carries the sCAR-4N1 expression cassette.
The oncolytic adenovirus portability external source antioncogene expression cassette that described method makes up.
The construction process of the oncolytic adenovirus of described target CD47 Positive Leukemic Cells, it may further comprise the steps:
1) expression cassette of structure sCAR-4N1;
2) enzyme cuts back to close the expression cassette of sCAR-4N1, is inserted into the corresponding restriction enzyme site of pXC2-sp-E1A plasmid, obtains the pXC2-sCAR-4N1-sp-E1A plasmid;
3) the pXC2 plasmid is introduced restriction enzyme site in situ behind deletion E1B, inserts external source antioncogene expression cassette structure and obtains pXC2-△ E1B-gene plasmid; Cut connection by enzyme the sCAR-4N1-sp fragment on the pXC2-sCAR-4N1-sp-E1A plasmid is replaced on the pXC2-△ E1B-gene plasmid, make up and obtain pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid;
4) pXC2-sCAR-4N1-sp-E1A plasmid and pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid respectively with pBHGE3 skeleton plasmid cotransfection packing cell, in packing cell, finish the assembling of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene;
5) separation and purification oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene.
Described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are the condition replication type adenovirus that is copied by the control of tomour specific promotor.
Described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are the condition replication type adenovirus that obtains by the deletion virogene.
Described external source antioncogene comprises one or more among IL-24, TRAIL, MnSOD, smac, p53, the PPA.
Described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are for the preparation of antitumor drug.
But described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene and oncolytic adenovirus Ad-sCAR-IL3-sp-E1A and/or oncolytic adenovirus Ad-sCAR-IL3-sp-E1A-△ E1B-gene combined preparation antitumor drug.
Embodiment 1
1) expression cassette of structure sCAR-4N1
Except specifying, the present invention can adopt this area routine techniques.
Make up the pQE30-sCAR-4N1 plasmid.Be 2 PCR method of template amplification sCAR-4N1 fusion gene with pQE30-sCAR-PPA plasmid (Li et al. 2009. Int J hematol. 89:611-617), the PCR upstream primer is 5 '-cccaagcttatggcgctcctgctgtgctt-3 ' (primer 1 for the first time, SEQ ID NO:1), downstream primer is 5 '-tcacaacataaaagcgggatccaggggcg-3 ' (primer 2, SEQ ID NO:2), for the second time PCR with the first time PCR product be template, upstream primer is 5 '-cccaagcttatggcgctcctgctgtgctt-3 ' (primer 1), downstream primer is 5 '-ccgaagctt ttaggatccaggggcggaag-3 ' (primer 3, SEQ ID NO:3).The pcr amplification product two ends have for the second time
HindIII restriction enzyme site, the method for cutting connection through enzyme are connected into pQE30 plasmid (available from German Qiagen company)
HindThe III site forms pQE30-sCAR-4N1, and this plasmid also can be used for the prokaryotic expression of sCAR-4N1.Utilize the pQE30-sCAR-4N1 plasmid, the sCAR-4N1 fusion gene can pass through
HindThe III enzyme cuts back to close.
The structure of pMD18-T simple-CMV-SV40 Poly A plasmid.This plasmid is made up by pMD18-T simple plasmid (available from Dalian TaKaRa biotech company) and pCA13 plasmid (available from Canadian Microbix Biosystem Inc company) and forms, purpose is that the CMV-SV40 Poly A expression cassette on the pCA13 plasmid is inserted pMD18-T simple plasmid, interstitial granules pMD18-T simple in the formation-CMV-SV40 Poly A, the convenient foreign gene that inserts.Utilize the PCR method to reclaim expression cassette on the pCA13 plasmid, upstream primer is 5 '-
GtcgacTaattccctggcattatgc-3 ' (primer 4, SEQ ID NO:4); Downstream primer is 5 '-
GtcgacAcgatccagacatgataag-3 ' (primer 5, SEQ ID NO:5).574 bp CMV-SV40 Poly A fragments and the pMD18-T simple carrier of PCR gained are connected to form pMD18-T simple-CMV-SV40 Poly A plasmid.
The structure of pMD18-T simple-CMV-sCAR-4N1-SV40 Poly A plasmid.With
HindThe III enzyme is cut the pQE30-sCAR-4N1 plasmid and is reclaimed the sCAR-4N1 fragment; the method of utilizing enzyme to cut connection is connected into pMD18-T simple-CMV-SV40 Poly A with the sCAR-4N1 fragment, namely forms pMD18-T simple-CMV-sCAR-4N1-SV40 Poly A plasmid.
2) enzyme cuts back to close CMV-sCAR-4N1-SV40 Poly A expression cassette fragment from pMD18-T simple-CMV-sCAR-4N1-SV40 Poly A plasmid plasmid, and expression cassette is connected into the pXC2-sp-E1A plasmid, namely forms the pXC2-sCAR-4N1-sp-E1A plasmid.Survivin promotor (sp) fragment can be utilized
SnaBI and
XhoThe I restriction enzyme site connects into pXC2 plasmid (available from Canadian Microbix Biosystem Inc company), namely forms pXC2-sp-E1A, this plasmid
XhoThe I site is used for the insertion of expression cassette.
3) the pXC2 plasmid is introduced restriction enzyme site in situ and is obtained pXC2-△ E1B plasmid behind deletion E1B, and insertion external source antioncogene expression cassette then makes up and obtains pXC2-△ E1B-gene plasmid; Cut connection by enzyme the sCAR-4N1-sp fragment on the pXC2-sCAR-4N1-sp-E1A plasmid is replaced on the pXC2-△ E1B-gene plasmid, make up and obtain pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid; (above-mentioned plasmid construction step as shown in Figure 1)
Be example with pXC2-sCAR-4N1-sp-E1A-△ E1B-IL24, the IL-24 expression cassette inserted pXC2-△ E1B plasmid namely form pXC2-△ E1B-IL24 plasmid.The pXC2-sCAR-4N1-sp-E1A plasmid can be recombinated with pXC2-△ E1B-IL24 plasmid, forms pXC2-sCAR-4N1-sp-E1A-△ E1B-IL24 plasmid.Utilize
NheI and
AleI double digestion shuttle plasmid pXC2-sCAR-4N1-sp-E1A reclaims small segment, and this fragment contains sCAR-4N1 expression cassette and sp promotor.Plasmid pXC2-△ E1B-IL24 uses equally
NheI and
AleThe I double digestion reclaims big fragment.Two fragments form pXC2-sCAR-4N1-sp-E1A-△ E1B-IL24 plasmid by connecting.
4) pXC2-sCAR-4N1-sp-E1A plasmid and pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid respectively with pBHGE3 skeleton plasmid cotransfection packing cell, in packing cell, finish the assembling of oncolytic adenovirus Ad-sCAR-4N1-sp-E1A and oncolytic adenovirus Ad-sCAR-4N1-sp-E1A-△ E1B-gene; Separation and purification oncolytic adenovirus Ad-sCAR-4N1-sp-E1A and oncolytic adenovirus Ad-sCAR-4N1-sp-E1A-△ E1B-gene.
PXC2-sCAR-4N1-sp-E1A plasmid and pXC2-sCAR-4N1-sp-E1A-△ E1B-IL24 plasmid can be respectively and the common transfection packing cell of adenovirus skeleton plasmid pBHGE3 plasmid, virus plaque appearred in 10-14 days, take turns the virus plaque purifying through 3, can not contained oncolytic adenovirus Ad.sCAR-4N1-sp-E1A or Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 that wild-type virus pollutes.
The evaluation of oncolytic adenovirus.Extract viral DNA, utilize PCR method to identify.The upstream primer of identifying sCAR-4N1 is 5 '-atggcgctcctgctgtgctt-3 ' (primer 6, SEQ ID NO:6), downstream primer is 5 '-tcactgttgagcctgcg-3 ' (primer 7, SEQ ID NO:7), as correctly amplifying the band of 1137 bp.Identify the upstream primer that whether contains wild-type virus be 5 '-cgcgggaaaactgaataaga-3 ' (primer 8, SEQ ID NO:8), downstream primer is 5 '-accgccaacattacagagtcg-3 ' (primer 9, SEQ ID NO:9), oncolytic adenovirus can amplify the band of 2300bp, and wild-type virus can amplify the band of 500bp.
The genome structure of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 as shown in Figure 2.
5) detect oncolytic adenovirus in external fragmentation effect to CD47+ leukemia cell
CD47+ leukemia cell K562 is with 2x10
4/ hole is laid on 96 orifice plates, is grouped as follows: PBS, Ad.sp-E1A, Ad.4N1 (abbreviation of Ad.sCAR-4N1-sp-E1A) and Ad.4N1-dBIL24 (abbreviation of Ad.sCAR-4N1-sp-E1A-△ E1B-IL24).If the dose gradient of oncolytic adenovirus is 20,50,100,150,200MOI.Act on mtt assay detection cell survival rate after 96 hours, data are to represent (Fig. 3 A) with respect to the percentage of PBS group.In addition, with identical grouping, the oncolytic adenovirus fixed dosage is under the 150MOI, and action time, gradient was made as 24,48,72,96 hours, and mtt assay detects cell survival rate, and data are to represent (Fig. 3 B) with respect to the percentage of PBS group.The result shows that the cells in vitro propagation inhibition ability of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 obviously is better than Ad.sp-E1A, and Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 obviously is better than Ad.sCAR-4N1-sp-E1A.
In the same way CD47+ leukemia cell HL-60 is carried out above-mentioned processing, also obtain similar results (Fig. 4 A, Fig. 4 B).
6) detect oncolytic adenovirus to the tumor killing effect of leukemia cell mouse transplanted tumor
CD47+ leukemia cell K562 subcutaneous transplantation BALB/c nude nude mice treats that tumour grows to 100m
3Carry out the animal grouping about size, comprise PBS group, Ad.sp-E1A group, Ad.4N1 (abbreviation of Ad.sCAR-4N1-sp-E1A) group and Ad.4N1-dBIL24 (abbreviation of Ad.sCAR-4N1-sp-E1A-△ E1B-IL24) group, carry out virus injection immediately, divide and carry out total dose 2,x10 4 times
9Pfu.The injection finish after every 4 staggering amount tumour sizes.The result shows that than all the other each groups, oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-IL24 has significantly suppressed the growth (Fig. 5) of K562 cell in nude mouse.
Embodiment 2
1) structure of the expression cassette of sCAR-4N1 is with embodiment 1.
2) structure of plasmid pXC2-sCAR-4N1-sp-E1A is with embodiment 1.
3) the pXC2 plasmid is introduced restriction enzyme site in situ and is obtained pXC2-△ E1B plasmid behind deletion E1B, inserts external source antioncogene expression cassette structure and obtains pXC2-△ E1B-gene plasmid; Cut connection by enzyme the sCAR-4N1-sp fragment on the pXC2-sCAR-4N1-sp-E1A plasmid is replaced on the pXC2-△ E1B-gene plasmid, make up and obtain pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid;
Be example with pXC2-sCAR-4N1-sp-E1A-△ E1B-TRAIL, construction step as shown in Figure 1.
Insert the TRAIL expression cassette at plasmid pXC2-△ E1B, namely constitute pXC2-△ E1B-TRAIL plasmid.The pXC2-sCAR-4N1-sp-E1A plasmid can be recombinated with plasmid pXC2-△ E1B-TRAIL, forms pXC2-sCAR-4N1-sp-E1A-△ E1B-TRAIL plasmid.Utilize
NheI and
AleI double digestion shuttle plasmid pXC2-sCAR-4N1-sp-E1A reclaims small segment, and this fragment contains sCAR-4N1 expression cassette and sp promotor.Plasmid pXC2-△ E1B-TRAIL uses equally
NheI and
AleThe I double digestion reclaims big fragment.Two fragments form pXC2-sCAR-4N1-sp-E1A-△ E1B-TRAIL plasmid by connecting.
4) pXC2-sCAR-4N1-sp-E1A-△ E1B-TRAIL plasmid and pBHGE3 skeleton plasmid cotransfection packing cell are finished the assembling of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-TRAIL in packing cell; Separation and purification oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-TRAIL.The authentication method of oncolytic adenovirus is the same.
In like manner, by inserting MnSOD at pXC2-△ E1B plasmid, smac, p53 or PPA(Rhizome of Pedate Pinellia lectin) expression cassette just can construct pXC2-△ E1B-MnSOD, pXC2-△ E1B-smac, pXC2-△ E1B-p53 or pXC2-△ E1B-PPA plasmid, these plasmids are by aforesaid method and the reorganization of pXC2-sCAR-4N1-sp-E1A plasmid, can construct pXC2-sCAR-4N1-sp-E1A-△ E1B-MnSOD, pXC2-sCAR-4N1-sp-E1A-△ E1B-smac, pXC2-sCAR-4N1-sp-E1A-△ E1B-p53 or pXC2-sCAR-4N1-sp-E1A-△ E1B-PPA plasmid, they respectively with pBHGE3 skeleton plasmid cotransfection packing cell, in packing cell, finish oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-MnSOD, Ad.sCAR-4N1-sp-E1A-△ E1B-smac, the assembling of Ad.sCAR-4N1-sp-E1A-△ E1B-p53 or Ad.sCAR-4N1-sp-E1A-△ E1B-PPA.The authentication method of oncolytic adenovirus is the same.
The present invention is not limited to above embodiment; some is based on technical scheme modification of the present invention; such as utilizing the different above-mentioned oncolytic adenovirus of middle plasmid construction; or carry sCAR-4N1 and external source antioncogene expression cassette with the oncolytic adenovirus of other type; or replace above-mentioned external source antioncogene with other external source antioncogene; or utilize different oncolytic adenovirus packaging systems to pack above-mentioned oncolytic adenovirus, all should think within protection scope of the present invention.
SEQUENCE LISTING
＜110〉Institutes Of Technology Of Zhejiang
＜120〉oncolytic adenovirus construction process of target CD47 Positive Leukemic Cells and uses thereof
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<170> PatentIn version 3.5
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Claims (6)
1. the construction process of the oncolytic adenovirus of a target CD47 Positive Leukemic Cells is characterized in that, the oncolytic adenovirus that described method makes up carries the sCAR-4N1 expression cassette or oncolytic adenovirus carries external source antioncogene expression cassette; Concrete construction process comprises the steps:
1) expression cassette of structure sCAR-4N1;
2) enzyme cuts back to close the expression cassette of sCAR-4N1, is inserted into the corresponding restriction enzyme site of pXC2-sp-E1A plasmid, obtains the pXC2-sCAR-4N1-sp-E1A plasmid;
3) the pXC2 plasmid is introduced restriction enzyme site in situ behind deletion E1B, inserts external source antioncogene expression cassette structure and obtains pXC2-△ E1B-gene plasmid; Cut connection by enzyme the sCAR-4N1-sp fragment on the pXC2-sCAR-4N1-sp-E1A plasmid is replaced on the pXC2-△ E1B-gene plasmid, make up and obtain pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid;
4) pXC2-sCAR-4N1-sp-E1A plasmid and pXC2-sCAR-4N1-sp-E1A-△ E1B-gene plasmid respectively with pBHGE3 skeleton plasmid cotransfection packing cell, in packing cell, finish the assembling of oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene;
5) separation and purification oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene.
2. the construction process of the oncolytic adenovirus of target CD47 Positive Leukemic Cells as claimed in claim 1, it is characterized in that described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are the condition replication type adenovirus that is copied by the control of tomour specific promotor.
3. the construction process of the oncolytic adenovirus of target CD47 Positive Leukemic Cells as claimed in claim 1, it is characterized in that described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are the condition replication type adenovirus that obtains by the deletion virogene.
4. the construction process of the oncolytic adenovirus of target CD47 Positive Leukemic Cells as claimed in claim 1 is characterized in that, described external source antioncogene comprises one or more among IL-24, TRAIL, MnSOD, smac, p53, the PPA.
5. the purposes of the oncolytic adenovirus of the target CD47 Positive Leukemic Cells that makes up of the method for claim 1, it is characterized in that described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene are for the preparation of antitumor drug.
6. purposes as claimed in claim 5, it is characterized in that described oncolytic adenovirus Ad.sCAR-4N1-sp-E1A and/or oncolytic adenovirus Ad.sCAR-4N1-sp-E1A-△ E1B-gene and oncolytic adenovirus Ad-sCAR-IL3-sp-E1A and/or oncolytic adenovirus Ad-sCAR-IL3-sp-E1A-△ E1B-gene combined preparation antitumor drug.
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| CN106190992A (en) * | 2015-04-30 | 2016-12-07 | 中国科学院上海生命科学研究院 | The construction method of cancer target gene-oncolytic adenovirus |
| CN108424461A (en) * | 2017-02-14 | 2018-08-21 | 亘喜生物科技（上海）有限公司 | CD47-CAR-T cells |
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| CN102747046A (en) * | 2012-06-20 | 2012-10-24 | 浙江理工大学 | Construction method and application of oncolytic adenovirus targeting CD123 positive leukemia cell |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106190992A (en) * | 2015-04-30 | 2016-12-07 | 中国科学院上海生命科学研究院 | The construction method of cancer target gene-oncolytic adenovirus |
| CN108424461A (en) * | 2017-02-14 | 2018-08-21 | 亘喜生物科技（上海）有限公司 | CD47-CAR-T cells |
| CN108424461B (en) * | 2017-02-14 | 2023-03-31 | 亘喜生物科技（上海）有限公司 | CD47-CAR-T cells |
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