CN1401778A - Recombinant of viral vector and human tumor suppressor gene, and use thereof - Google Patents
Recombinant of viral vector and human tumor suppressor gene, and use thereof Download PDFInfo
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Abstract
A recombinant adenovirus p53 is constructed from adenovirus vector and human p53 gene expression cassette by the homologous recombination of adenovirus vector with human p53 gene in E coli cell. Said human p53 gene expression cassette is a characteristic sequence of promoter-p53cDNA-polyadenine nucleotide. Said recombinant adenovirus p53 can be used to prepare clinic gene medicine for preventing and treating cancers.
Description
Technical field
The present invention relates to genetic engineering technique, more particularly relate to a kind of recombinant chou that is built into by human tumor suppressor gene p53 and adenovirus carrier recombination sequence.
Background technology
Along with the development of the progress of Protocols in Molecular Biology, particularly genetic engineering technique, the gene therapy of malignant tumour becomes the focus of people's research.At present, the whole world has 600 multinomial gene therapy schemes and gets permission to enter clinical trial, 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 do not have the meaning of any treatment disease, no clinical value; And,, and do not possess practical clinical value in target cell because the difficulty that imports target cell and express only has the potential therapeutic action as can be used for various therapeutic purpose genes.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, and the process complexity wastes time and energy.Use the new technology that in prokaryotic cell prokaryocyte (intestinal bacteria), realizes homologous recombination, vector construction hexose transport protein then can effectively address the above problem.
The gene transfer vector that lacks specificity, target and high efficiency is a difficult problem of therapy of tumor always.At present, the carrier that is used for gene therapy research 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 and also can infect non-division stage cell, have safety, pathogenic advantage such as low.But also there is weak point, 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, when allogenic gene is carried in adenovirus carrier E1 or E3 disappearance district, can realize the expression of the long period of goal gene, and can reduce its immunogenicity; Retroviral vector can carry allogenic gene and be integrated in the target cell genome and to realize that goal gene stablizes persistent expression, but the external breeding titre of retrovirus is low, transfection efficiency is low, only infects the division stage cell.To chromosomal random integration, carcinogenic danger is arranged; All there are different pros and cons in other virus vector that can be used for transgenosis with non-virus carrier.
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 the recombinant chou of a kind of virus vector and human tumor suppressor gene, make human tumor suppressor gene and virus vector constitute fusion sequence, make it breeding in the specific cells of genetic engineering modified mistake, produce, and can directly in eukaryotic cell, express, reach prevention or/and the purpose of treatment tumour.
The present invention also aims to provide the preparation method of this recombinant chou and be used to prepare prevention or/and the application of medicine for treating tumor thing.
For achieving the above object, the invention provides the recombinant chou of a kind of virus vector and human tumor suppressor gene, this recombinant chou is that the virus vector by the dna clone technique construction is combined with the human tumor suppressor gene expression cassette, 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 expressing tumor arrestin.
The carrier of this recombinant chou 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, and most preferably carrier is an adenovirus carrier.
Described human tumor suppressor gene can be to have in the gene of tumor inhibition effect any, its most preferably gene be the p53 gene.
This recombinant chou is by adenovirus carrier and p53 is gene constructed forms, be defined as the recombinant P 53 adenovirus body, its fusion sequence is: adenovirus 5 genome sequences right side-ATGTTTACCGCCACACTCGCAGGGTCTGCACCTGGTGCGGGTCTCATCGTACCTCA GCACCTTCCAGATC
70TCTGACATGCGATGTCGACTCGACTGCTTCGCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTTGGTACCGAGCTCGGATCCCG
523CTAGAGCCACCGTCCAGGGAGCAGGTAGCTGCTGGGCTCCGGGGACACTTTGCGTT CGGGCTGGGAGCGTCTTTCCACGACGGTGACACGCTTCCCTGGATTGGCAGCCAGA CTGCTTTCCGGGTCACTGCC
655ATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGA
1837CATTCTCCACTTCTTGTTCCCCACTGACAGCCTCCCACCCCCATCTCTCCCTCCCCTGCCATTTTGGGTTTTGGGTCTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGGGCTCCACTGAACAAGTTGGCCTGCACTGGTGTTTTGTTGTGGGGAGGAGGATGGGGAGTAGGACATACCAGCTTAGATTTTAAGGTTTTTACTGTGAGGGATGTTTGGGAGATGTAAGAAATGTTCTTGCAGTTAAGGGTTAGTTTACAATCAGCCACATTCTAGGTAGGGGCCACTTCACCGTACTAACCAGGGAAGCTGTCCCTCACTGTTGAATTTTCTCTAACTTCAAGGCCCATATCTGTGAAATGCTGGATTTGCCCTACCTCGGAATGCTGGCATTTGCACCTACCTCACAGAGTGCATTGTGAGGGTT
2297AATGAAATAATGTACATCTGGCCTTGAAACCACCTTTTATTACATGGGGTCTAGCGGGATCCACTAGTAACGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGGGGGATCCCCACGCTAGAGCT
2733GACTATAATAATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGAGAAAA ACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATA TTA
2848-adenovirus 5 genome sequences left sides is wherein: 1. adenovirus 5 genom sequences (Genbank No:NC_001406) 2.1-70 is seen in adenovirus 5 genome sequence right sides and adenovirus 5 genome sequences left side: coded sequence 6.1838-2733:3 ' end non-translational region (wherein being poly-A tail poly A since the 2298) 7.2734-2848 of adenovirus right arm (70 bit bases are positioned at 3328 of adenovirus 5 genome sequence forwards) the sarcoma viral LTR of 3.71-523:Rous (promoter) 4.524-655:5 ' end non-translational region 5.656-1837:p53 gene: adenovirus left side arm (2734 bit bases are positioned at adenovirus 5 genome sequence forwards 452 bit bases)
The expression casette of this recombinant chou is the characteristic sequence by promotor-p53cDNA-poly adenine nucleotide constitutes, its upstream is arbitrary eukaryotic cell promotor, prokaryotic cell prokaryocyte promotor or viral promotors, and the downstream is the poly adenine nucleotide of any eukaryotic gene.
Recombinant chou of the present invention obtains by the following method, recombinant viral vector homologous recombination in prokaryotic cell prokaryocyte obtains, at first be that adenovirus and plasmid pGT-1 (containing adenovirus both sides inverted repeats) homologous recombination in intestinal bacteria obtains recombinant chou pGT-2, again with artificial constructed " adenovirus right arm/promotor-p53cDNA-poly adenine nucleotide/adenovirus left side arm " homologous recombination acquisition recombinant chou pGT-3 in intestinal bacteria, with after the protokaryon plasmid sequence is removed in restriction endonuclease PacI linearizing, obtain the recombinant P 53 adenovirus body.
In fact, this recombinant chou can the mode with homologous recombination obtain in any prokaryotic cell prokaryocyte.
According to such scheme,, introduce the PacI restriction enzyme site respectively with the LTR sequence of pcr amplification adenovirus 5 both sides.The LTR sequence of both sides all is cloned in the pUC18 carrier, constitutes recombinant vectors pGT-1; The pGT-1 carrier and the adenovirus 5 genome cotransfection e. coli strains BJ5183 that make up (are matched the preservation of hundred promise companies, preserve number: P-e012), make adenovirus 5 genomes and pGT-1 that homologous recombination take place, the positive-virus clone cuts evaluation through amplification, PCR screening and enzyme, obtains to contain adenovirus 5 complete genomic recombinant vectors pGT-2.
With 5 ' ATGGAGGAGCCGCAGTCAGATC and 5 ' ATATCTGCAGAATTCCAGCAC as primer, by pcr amplification human tumor supressor p53 gene, the total length p53 gene of amplification (contain 5 ' and 3 ' end non-translational region sequence) is cloned into protokaryon plasmid pUC19, carries out sequence verification.Subsequently, PCR increase respectively the LTR sequence (containing promotor) of RSV (rous sarcoma virus), PA sequence and the adenovirus E 1 region sequence of BGH, and introduce linker sequence, sequence verification respectively in a side.Carry out PCR when reaction once more, LTR and PA sequence are spliced to respectively near 5 of p53 gene ' and 3 ' end.Adenovirus E 1 district and upstream sequence thereof are spliced to the outermost of p53 gene respectively, constitute p53 complex gene (see figure 1).
With recombinant vectors pGT-2 and the p53 complex gene cotransfection e. coli strains BJ5183 that builds, make the two that homologous recombination take place.The same, positive colony is cut evaluation through amplification, PCR screening and enzyme.Obtain recombinant vectors pGT-3, contain adenovirus 5 most of sequences (its E1 district and upstream portion sequence are replaced by the p53 expression casette) in this carrier.Recombinant vectors pGT-3 is through the PacI linearization for enzyme restriction, removes the carrier sequence that derives from pUC18, and (the hundred promise companies that match are frozen, preserve number: E-393) cultivate for rotaring redyeing 293 cell.Become to contain the human tumor suppressor gene p53 of the promotor manipulation of adenovirus cis activation sequences (cis-acting sequence) and LTR in the cell internal packing.Set up into the transfection efficiency height, workable, by the recombinant P 53 adenovirus body of single promotor control.
This recombinant P 53 adenovirus body has following characteristics:
It is to be made of adenovirus carrier and the artificial expression cassette two portions of p53 gene,
1, constructional feature: its essence is the recombinant adenovirus body of a work, is different from existing chemical synthetic drug, Chinese medicine, genetically engineered drug, is the expression in vivo of directly realizing goal gene, and the biologic activity height can effectively reach therapeutic action; Adenovirus carrier can carry bigger gene, transfection efficiency height, can prepare the virion that height is tired, host range is wide, and security is good, and is pathogenic low, especially the adenovirus carrier immunogenicity after reconstruction descends greatly, makes goal gene be easy to stable in body, the lasting expression; The artificial expression cassette of p53 gene is with adenovirus carrier list promotor direct regulation and control p53 expression of gene, and complete polyadenylic acid tailing signal is arranged, thereby constitute a complete expression cassette (expression cassette), adjustable p53 gene efficiently expresses in target cell.
2, application characteristic: this recombinant P 53 adenovirus body is the wide spectrum antitumor drugs, has the therapeutic action to various malignant tumours.II phase clinical experiment shows that ten kinds of tumors such as the correct neck squamous cell carcinoma of this recombinant P 53 adenovirus body, lung cancer have the obvious treatment effect; This recombinant P 53 adenovirus body has the unique effect that prophylaxis of tumours takes place.I clinical trial phase and postoperative were followed up a case by regular visits to and are shown that this recombinant P 53 adenovirus body can prevent the postoperative recurrence of tumour patients such as laryngocarcinoma in 3 years, played the effect of tumor vaccine.
Utilize recombinant P 53 adenovirus body of the present invention can prepare the medicine for the treatment of various malignant tumours, and can prepare the generation of prophylaxis of tumours and the medicine of the tumor regrowth behind the excision.
Recombinant P 53 adenovirus body of the present invention also can be used for preparing the medicine that carries out injection in intravenous injection, intra-arterial injection, intratumor injection, intramuscular injection, subcutaneous injection, organ injection and chest, the ascites.
The present invention utilizes prepared recombinant P 53 adenovirus body, and first transfection is cultivated, bred in the specific cells of genetic engineering modified mistake, and concentrated and purified one-tenth can be used for clinical recombinant adenovirus p53 anti-cancer injection.
Wherein the present invention tests used 293 clones (ATCC CRL-1573, the 32nd generation, on June 13rd, 1997 ordered from ATCC) derive from and transform the human embryo kidney epithelial cell through 5 type adenovirus (Ad5) DNA and obtain, contain 11% genome (comprising E1a) of Ad5 5 ' end.This cell is highly permission to the infection and the growth of adenovirus.
Use this recombinant P 53 adenovirus body in 12 examples, late period patients with laryngeal carcinoma carry out in I phase clinical experiment, also followed up a case by regular visits to 31-36 month treatment back.The result shows that this recombinant P 53 adenovirus body is safe in utilization, and whole 12 routine patients do not have tumor recurrence.At present, the middle and advanced stage patients with laryngeal carcinoma has only 44% 3 year lifetime clinically, 6-12 month the recurrence rate about 20% in operation back.Experimental result of the present invention shows that this recombinant P 53 adenovirus body has the dual function of treatment and prophylaxis of tumours recurrence.Carry out II phase clinical experiment since calendar year 2001, shown the good curing effect.The result of embodiment accompanying drawing 9 to 13 shows that some use recombinant P 53 adenovirus body of the present invention still to have the good curing effect to the invalid patient of conventional treatments (chemotherapy and radiation).
Contribution of the present invention is that it utilizes human tumor suppressor gene p53 can suppress the characteristics of kinds of tumor cells growth, and it is cloned into adenovirus E 1
-Strain by knurl body injection, makes adenovirus effectively the p53 gene be imported tumor tissues, give expression to P53 albumen after, suppress the growth of tumor tissues, make tumour cell retarded growth or apoptosis occur, thereby reach the purpose that suppresses tumour.This invention has also solved owing to P53 albumen transformation period short (about 20 minutes), instability simultaneously, can't externally be prepared into the problem into the reorganization gene engineering product, has promptly realized the oncotherapy of p53 gene.
This recombinant P 53 adenovirus body utilizes adenovirus to carry human caused by tumor suppressor p 53, directly in tumour cell, express, thereby solved because P53 albumen instability, can't be with the external problem that is prepared into the recombination engineering product of engineered method.Carry out cancer therapy with adenovirus and adeno-associated virus, patient can continue, efficiently express P53 albumen in vivo, and on protein molecule modification program, all have the characteristic identical with eukaryote in the body as aspects such as protein phosphorylation, protein folding and multimerizations.Recombinant P 53 adenovirus body of the present invention can directly mediate the p53 gene and express in eukaryotic cell, that is to say that can directly import the solid tumor position allows its expression, makes curee self become one and can produce human tumor supressor P53 proteic " source ".This method realizes shifting in the body of external source p53 gene and efficiently expressing in patient's knurl body and reaches therapeutic purpose, makes the gene therapy of tumour and other disease become a reality.
Description of drawings
Fig. 1 is the building process synoptic diagram of recombinant P 53 adenovirus body of the present invention.
Fig. 2 is the technological line block diagram of recombinant P 53 adenovirus body of the present invention.
Fig. 3 is that the recombinant P 53 adenovirus body is after repeatedly going down to posterity, with 5 ' CCACGACGGTGACA CGCTTC and 5 ' CAAGCAAGGGTTCAAAGAC is primer, with p53cDNA is masterplate, and the agarose gel electrophoresis figure of the p53 gene that obtains by pcr amplification is to identify its stability.
Fig. 4 be the recombinant P 53 adenovirus body (the hundred promise companies that match are frozen, preserve number: No-1, down with) infected behind 293 cells 36 hours, through lysis, extract the agarose gel analysis result that viral DNA is identified through PCR.
Fig. 5 is that the recombinant P 53 adenovirus body infected behind 293 cells 36 hours, through lysis, extraction after Western blot analytical results.
Fig. 6 is the curve synoptic diagram of recombinant P 53 adenovirus body various dose to the lethal effect of Hep-2 cell.
Fig. 7 is the inhibiting curve synoptic diagram of recombinant P 53 adenovirus body to the growth of Hep-2 cell.
Fig. 8 is that the recombinant P 53 adenovirus body infects the light microscopic photo of Hep-2 cell after 36 hours.
Fig. 9 is the CT photo of recombinant P 53 adenovirus body to clinical nasopharyngeal cancer patient therapeutic action (the II phase is clinical).
Figure 10 is the CT photo of recombinant P 53 adenovirus body to clinical lung carninomatosis human therapy effect (the II phase is clinical).
Figure 11 is the CT photo of recombinant P 53 adenovirus body to clinical thyroid carcinoma patient effect (the II phase is clinical).
Figure 12 is the CT photo of recombinant P 53 adenovirus body to clinical cervical cancer patient effect (the II phase is clinical).
Figure 13 is the CT photo of recombinant P 53 adenovirus body to clinical esophagus cancer patient effect (the II phase is clinical).
Embodiment
The following example is to further explanation of the present invention and explanation, and the present invention is not constituted any limitation.
As depicted in figs. 1 and 2, make up recombinant P 53 adenovirus body and evaluation
1, the p53 gene cDNA complete sequence of having delivered, the synthetic two sections primers of design:
5 ' ATGGAGGAGCCGCAGTCAGATC and 5 ' ATATCTGCAGAATTCCAGCAC are as primer, and the linker sequence is introduced at two ends respectively.With the HeLa cell cDNA is template, obtains the human P 53 gene through PCR method amplification, and wherein, reaction conditions is: first circulation: 94 ℃ of sex change 4 minutes, and 58 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes; Each circulation later on: 94 ℃ of sex change 1 minute, 58 ℃ of annealing 1 minute, 72 ℃ were extended totally 30 circulations 2 minutes.Obtain a large amount of p53 genes thus, analyze with agarose gel electrophoresis, recovery obtains the p53 full length gene, cut the p53 gene with this enzyme again behind the fragment purification, be inserted in the pUC19 carrier that same enzyme cuts and check order, the base sequence that records the coding region with deduced amino acid (consistent) with GenBank Acc XM_058834, enzyme cuts back to close subsequently.
2, PCR reaction amplification LTR and PA sequence, primer is respectively: 5 ' TCTGACATGCGATGTCGACTCG, 5 ' CGGCAGTGACCCGGAAAGCAG; 5 ' TCACAGAGTGCATTGTGAGGG, 5 ' GCTCTAGCGTGGGGATCCC.Introduce the linker sequence respectively at 5 ' primer and 3 ' end primer.Under the same annealing conditions, pcr amplification LTR and PA sequence, purifying rear clone sequence verification.
3, PCR reacts the E1 sequence of the adenovirus that separately increases, and reaction conditions is the same, and the two ends primer is introduced Bam HI and Eco RI restriction enzyme site respectively, amplification back sequence verification.
4,1 calling sequence is carried out the PCR reaction with the two sequences of 2 gained respectively, reaction conditions is the same, obtains PCR splicing product LTR-p53-PA, further sequence verification.
5,3 calling sequences and 4 calling sequence LTR-p53-PA are bonding through T4 DNA splicing enzyme, obtain the p53 complex gene.
6, PCR reacts the adenovirus two ends IRT sequence that increases respectively, and reaction conditions is the same, respectively two sections sequence clones is constituted recombinant vectors pGT-1 in the pUC18 carrier after the sequence verification.
7, extract wild-type adenovirus 5 (ATCC-VR-5, adenopathy strain 75, titre: 10 (6.75) TCID (50)/ml) DNA, with recombinant vectors pGT-1 cotransfection intestinal bacteria BJ5183, hatched 30 minutes through 4 ℃, 42 ℃ of 50 seconds of heat-shocked, hatched 1 minute at 4 ℃ again, add 1ml LB nutrient solution 1 hour, change the engineering bacteria of incubation over to contain peace Bian penicillin agar culture plate, after 24 hours,, put into the clean culturing bottle that contains LB then with the single bacterial clone of sterilization toothpick picking, after 24 hours, extract plasmid according to a conventional method, with PacI cutting screening, the acquisition positive colony is pGT-2 (containing the Ad5 complete sequence).
8, with p53 complex gene and recombinant vectors pGT-2 cotransfection intestinal bacteria BJ5183, the same method is cultivated, screening and evaluation, obtain positive colony pGT-3, the p53 expression casette that contains adenovirus whole genome sequence and insertion, cut with the PacI enzyme again, make it linearizing, remove the carrier sequence that derives from pUC18.
9, with isolating positive plasmid linearizing after CsCl
2Purifying is through CaCl
2The method rotaring redyeing 293 cell.After 7 days, collecting cell, centrifugal 15 minutes of 1000rpm abandons supernatant, and cell is through 37 ℃ of-80 ℃ of freeze thawing, cracking three times, centrifugal 30 minutes of 4000rpm gets supernatant, abandons precipitation, and supernatant is through superinfection, amplicon virus, lytic virus in kind, supernatant is through CsCl
2Density gradient centrifugation, condition is: 4 ℃ of 60000rpm, 16 hours.Get recombinant adenovirus body decoupled band through No. 7 injection needless.Dialysed 4 hours in the N1H damping fluid with the SpectraMW6000 dialysis tubing, whole process keeps 4 ℃.Take out viral liquid, with the membrane filtration degerming of 0.25 μ m, packing.In-80 ℃ of preservations, a part is made plaque-forming assay and virion assay.
10, the structural stability of recombinant P 53 adenovirus body is identified, after repeatedly going down to posterity, extract its genomic dna, be primer with p53 gene two ends 5 ' CCACGACGGTGACACGCTTC and 5 ' CAAGCAAGGGTTCAAAGAC, carry out pcr amplification, agarose gel electrophoresis the results are shown in Figure 3; The adenovirus arm 5 ' TTT CTC AGG TGT TTT CCG C and the 5 ' CATCGT ACC TCA GCA CCT TC that reach the device both sides with the recombinant P 53 adenovirus body surface are primer, and the PCR qualification result is seen Fig. 4.Above result all shows recombinant P 53 adenovirus body Stability Analysis of Structures after repeatedly going down to posterity.
12, the p53 gene of recombinant P 53 adenovirus body mediation is identified in the expression of Hep-2 and H1299 cell.Infect 293 cells after 36 hours with the recombinant P 53 adenovirus body, the ordinary method lysing cell carries out western blot with the P53 protein specific antibody and analyzes, and the results are shown in Figure 5.
13, the recombinant P 53 adenovirus body is to the time and the dose relationship of Hep-2 cytosis.The recombinant P 53 adenovirus body infected the Hep-2 cell after 36 hours, studied its various dose and the lethal effect of different action times to the Hep-2 cell respectively.With the blue dyeing numeration of tongue phenol dead cell number, the results are shown in Figure 6 and Fig. 7.
The recombinant P 53 adenovirus body is to the tumor cytotoxicity effect:
Hep-2 cell with recombinant P 53 gene adenovirus ordinary method transfection cultivation.It is identical that each organizes cell count, and experiment divides following each group: 1. blank; 2.MOI 200; 3.MOI 150; 4.MOI 100; 5.MOI 50; 6.MOI 10.7.GFP?virus?MOI?200；8.GFP?virus?MOI?150。Continue to cultivate after the transfection 36 hours, om observation as seen in MOI＞50 o'clock, all can be seen the lethal effect of recombinant P 53 adenovirus body to the Hep-2 cell, and along with the increase lethal effect of dosage is more obvious.Apoptosis performances such as tangible pyknosis diminishes appear in the om observation cell, as shown in Figure 8.
The clinical treatment effect (nasopharyngeal carcinoma) of recombinant P 53 adenovirus body:
As shown in Figure 9, the recombinant P 53 adenovirus body carries out II phase clinical experiment (down together) in specified hospital of National Drug Administration, Fig. 9 is shown as a nasopharyngeal carcinoma patient at the CT photo of accepting reorganization recombinant P 53 adenovirus body treatment front and back, left side figure is the cancerous swelling piece before the treatment, right figure is the back of receiving treatment (lump dwindles 87%, occurs downright bad in companion's knurl).Have above 60 routine patients at present and accepted II phase clinical experiment, curative effect is conclusive.
The clinical treatment effect (lung cancer) of recombinant P 53 adenovirus body:
As shown in figure 10, II phase clinical experiment.The recombinant P 53 adenovirus body in II phase clinical experiment to the therapeutic action of lung cancer.Women left side adenocarcinoma of lung patient, left thoracic cavity hydrops, companion's hepatic metastases, bone shift and brain shifts.After repeatedly drawing liquid and chemotherapy, still have a large amount of hydrops to occur in the thoracic cavity.Patient accepted intrathoracic injection recombinant P 53 adenovirus body after one month, hydrops completely dissolve in the thoracic cavity.
The clinical treatment effect (thyroid carcinoma) of recombinant P 53 adenovirus body:
As shown in figure 11, II phase clinical experiment.The recombinant P 53 adenovirus body in II phase clinical experiment to the therapeutic action of thyroid carcinoma.Women's thyroid carcinoma patient, 1 year local recurrence of postoperative, upper right neck lymphatic metastasis, pathological diagnosis is low differentiated squamous-cell carcinomas, and is invalid after 1 year of treatment through radiotherapy-chemotherapy-combined with hyperthermia, throat oncothlipsis tracheae, expiratory dyspnea.Accept the treatment of recombinant P 53 adenovirus body subsequently, inject altogether 8 times, lump obviously dwindles (dwindling 48%).
The clinical treatment effect (cervical cancer) of recombinant P 53 adenovirus body:
As shown in figure 12, II phase clinical experiment.The recombinant P 53 adenovirus body in II phase clinical experiment to the therapeutic action of cervical cancer.Cervical cancer patient, pathological diagnosis is the squama cancer.After accepting the treatment (injecting altogether 8 times) of recombinant P 53 adenovirus body, lump obviously dwindles (about 91%), check after three months, lump completely dissolve.
Embodiment 7
The clinical treatment effect of recombinant P 53 adenovirus body (esophagus cancer transfer):
As shown in figure 13, II phase clinical experiment.The recombinant P 53 adenovirus body in II phase clinical experiment to the therapeutic action of esophagus cancer nodus lymphoideus transferring rate.Esophagus cancer left clavicle superior gluteal lymph node shifts the patient, and pathological diagnosis is the squama cancer.Invalid after radiotherapy, accept the treatment of recombinant P 53 adenovirus body after, inject altogether 8 times, lump obviously dwindles (dwindling 29%).
Claims (13)
1, the recombinant chou of a kind of virus vector and human tumor suppressor gene, it is characterized in that, this recombinant chou is that the virus vector by the dna clone technique construction combines with the human tumor suppressor gene expression cassette, 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 expressing tumor arrestin.
2, recombinant chou as claimed in claim 1 is characterized in that, the carrier of this recombinant chou can be any of dna virus or RNA viruses.
3, recombinant chou as claimed in claim 2 is characterized in that, the carrier of this recombinant chou is adenovirus carrier or the complex carrier that contains the adenovirus carrier sequence.
4, recombinant chou as claimed in claim 1 is characterized in that, described human tumor suppressor gene can be to have in the gene of tumor inhibition effect any.
5, recombinant chou as claimed in claim 4 is characterized in that, described human tumor suppressor gene is the p53 gene.
6, recombinant chou as claimed in claim 1, it is characterized in that, it is by adenovirus carrier and p53 is gene constructed forms, and its fusion sequence is: adenovirus 5 genome sequences right side-ATGTTTACCGCCACACTCGCAGGGTCTGCACCTGGTGCGGGTCTCATCGTACCTCA GCACCTTCCAGATC
70TCTGACATGCGATGTCGACTCGACTGCTTCGCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTTGGTACCGAGCTCGGATCCCG
523CTAGAGCCACCGTCCAGGGAGCAGGTAGCTGCTGGGCTCCGGGGACACTTTGCGTT CGGGCTGGGAGCGTCTTTCCACGACGGTGACACGCTTCCCTGGATTGGCAGCCAGA CTGCTTTCCGGGTCACTGCC
655ATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGA
1837CATTCTCCACTTCTTGTTCCCCACTGACAGCCTCCCACCCCCATCTCTCCCTCCCCTGCCATTTTGGGTTTTGGGTCTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGGGCTCCACTGAACAAGTTGGCCTGCACTGGTGTTTTGTTGTGGGGAGGAGGATGGGGAGTAGGACATACCAGCTTAGATTTTAAGGTTTTTACTGTGAGGGATGTTTGGGAGATGTAAGAAATGTTCTTGCAGTTAAGGGTTAGTTTACAATCAGCCACATTCTAGGTAGGGGCCACTTCACCGTACTAACCAGGGAAGCTGTCCCTCACTGTTGAATTTTCTCTAACTTCAAGGCCCATATCTGTGAAATGCTGGATTTGCCCTACCTCGGAATGCTGGCATTTGCACCTACCTCACAGAGTGCATTGTGAGGGTT
2297AATGAAATAATGTACATCTGGCCTTGAAACCACCTTTTATTACATGGGGTCTAGCGGGATCCACTAGTAACGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGGGGGATCCCCACGCTAGAGCT
2733GACTATAATAATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGAGAAAA ACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATA TTA
2848-adenovirus 5 genome sequences left sides is wherein: 1) adenovirus 5 genom sequences (Genbank No:NC_001406) 2 are seen in adenovirus 5 genome sequence right sides and adenovirus 5 genome sequences left side) 1-70: adenovirus right arm (70 bit bases are positioned at 3328 of adenovirus 5 genome sequence forwards) 3) the sarcoma viral LTR of 71-523:Rous (promoter) 4) 524-655:5 ' hold non-translational region 5) coded sequence 6 of 656-1837:p53 gene) 1838-2733:3 ' end non-translational region (wherein being poly-A tail poly A since 2298) 7) and 2734-2848: arm (2734 bit bases are positioned at adenovirus 5 genome sequence forwards 452 bit bases) on the left of the adenovirus
7, recombinant chou as claimed in claim 1 is characterized in that, the expression casette of this recombinant chou is the characteristic sequence by promotor-p53cDNA-poly adenine nucleotide constitutes.
8, recombinant chou as claimed in claim 7 is characterized in that, the upstream of described expression casette is arbitrary eukaryotic cell promotor, prokaryotic cell prokaryocyte promotor or viral promotors, and the downstream is the poly adenine nucleotide of any eukaryotic gene.
9, recombinant chou as claimed in claim 1, it is characterized in that, this recombinant chou homologous recombination in prokaryotic cell prokaryocyte obtains, at first be that adenovirus and plasmid pGT-1 (containing adenovirus both sides inverted repeats) homologous recombination in intestinal bacteria obtains recombinant chou pGT-2, again with artificial constructed " adenovirus right arm/promotor-p53cDNA-poly adenine nucleotide/adenovirus left side arm " homologous recombination acquisition recombinant chou pGT-3 in intestinal bacteria, with after the protokaryon plasmid sequence is removed in restriction endonuclease PacI linearizing, obtain the recombinant P 53 adenovirus body.
10, recombinant chou as claimed in claim 9 is characterized in that, this recombinant chou mode with homologous recombination in any prokaryotic cell prokaryocyte obtains.
11, recombinant chou as claimed in claim 1 is used to prepare the medicine for the treatment of various malignant tumours.
12, recombinant chou as claimed in claim 1 is used to prepare the generation of prophylaxis of tumours and the medicine of the tumor regrowth behind the excision.
13, recombinant chou as claimed in claim 1 is used to prepare the medicine that carries out injection in intravenous injection, intra-arterial injection, intratumor injection, intramuscular injection, subcutaneous injection, organ injection and chest, the ascites.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2004078987A1 (en) * | 2003-03-06 | 2004-09-16 | Zhaohui Peng | A recombinant constructed by a virus vector and a human tumor suppressor gene and its use |
| WO2004104204A1 (en) * | 2003-05-10 | 2004-12-02 | Zhaohui Peng | RECOMBINANT GENE MEDICINE OF ADENOVIRUS VECTOR AND GENE p53 FOR TREATING PROLIFERATIVE DISEASES |
| EP1842921A1 (en) * | 2005-01-26 | 2007-10-10 | Zhaohui c/o SiBiono Co. Peng | Recombined adenovirus p53 preparation for treating tumor |
| CN104094120A (en) * | 2011-12-08 | 2014-10-08 | 凡弗3基因组有限公司 | Mdm2-containing double minute chromosomes and methods therefore |
| CN105755043A (en) * | 2016-04-12 | 2016-07-13 | 高贵 | Double-copy human p53 gene recombinant adenovirus and preparation method thereof |
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2002
- 2002-05-08 CN CNB021152284A patent/CN1177057C/en not_active Expired - Lifetime
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| WO2004078987A1 (en) * | 2003-03-06 | 2004-09-16 | Zhaohui Peng | A recombinant constructed by a virus vector and a human tumor suppressor gene and its use |
| AU2004240968B2 (en) * | 2003-05-10 | 2008-04-17 | Zhaohui Peng | Recombinant gene medicine of adenovirus vector and gene p53 for treating proliferative diseases |
| JP2007504274A (en) * | 2003-05-10 | 2007-03-01 | ペン、ジャオフイ | Recombinant gene drug of adenoviral vector and P53 gene for treating proliferative diseases |
| CN1327899C (en) * | 2003-05-10 | 2007-07-25 | 彭朝晖 | Gene recombined medicine of adenovirus carrier and p53 gene for treating proliferative diseases |
| WO2004104204A1 (en) * | 2003-05-10 | 2004-12-02 | Zhaohui Peng | RECOMBINANT GENE MEDICINE OF ADENOVIRUS VECTOR AND GENE p53 FOR TREATING PROLIFERATIVE DISEASES |
| JP4695086B2 (en) * | 2003-05-10 | 2011-06-08 | ペン、ジャオフイ | Recombinant gene drug of adenoviral vector and P53 gene for treating proliferative diseases |
| EP1842921A1 (en) * | 2005-01-26 | 2007-10-10 | Zhaohui c/o SiBiono Co. Peng | Recombined adenovirus p53 preparation for treating tumor |
| EP1842921A4 (en) * | 2005-01-26 | 2008-02-13 | Zhaohui Peng | Recombined adenovirus p53 preparation for treating tumor |
| CN104094120A (en) * | 2011-12-08 | 2014-10-08 | 凡弗3基因组有限公司 | Mdm2-containing double minute chromosomes and methods therefore |
| CN104094120B (en) * | 2011-12-08 | 2017-04-26 | 凡弗3基因组有限公司 | Mdm2-containing double minute chromosomes and methods therefore |
| US10774384B2 (en) | 2011-12-08 | 2020-09-15 | Five3 Genomics, Llc | MDM2-containing double minute chromosomes and methods therefore |
| US10961586B2 (en) | 2011-12-08 | 2021-03-30 | Five3 Genomics, Llc | MDM2-containing double minute chromosomes and methods therefore |
| CN105755043A (en) * | 2016-04-12 | 2016-07-13 | 高贵 | Double-copy human p53 gene recombinant adenovirus and preparation method thereof |
| CN105755043B (en) * | 2016-04-12 | 2019-03-15 | 高贵 | A kind of pair of copy Human p53 gene recombined adhenovirus and preparation method thereof |
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| CN1177057C (en) | 2004-11-24 |
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