CN102002483B - Hematopoietic targeted helper adenovirus and application thereof - Google Patents
Hematopoietic targeted helper adenovirus and application thereof Download PDFInfo
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Abstract
The invention relates to a hematopoietic targeted helper adenovirus and application thereof, belonging to the field of biotechnologies and gene therapy. The invention comprises the construction and the preparation of the hematopoietic targeted helper adenovirus, the preparation, the amplification and the purification of helper-dependent adenoviral vectors (HD-Ad) and the verification of hematopoietic targeting function. A packaging signal carried by the helper adenovirus is an incomplete packaging signal, both ends of the packaging signal contain loxp sequences, homologous recombination can be carried out under the action of a Cre recombinase, and the packaging signal is cut off to further reduce the production of helper adenovirus and improve the yield and the quality of the target virus. A skeleton plasmid of the helper adenovirus is pAd5/F11p, and a fiber top ball changed from the skeleton plasmid can enable the helper adenovirus to have hematopoietic targeting characteristic. The HD-Ad prepared by using the helper adenovirus has the particle characteristics of helper adenovirus and can effectively target hematopoietic cells. Thus, the invention provides an experiment basis and a theoretical basis for the application of HD-Ad in hematopoietic cell gene therapy.
Description
Technical field
The present invention relates to biotechnology and field of gene, be specially a kind of structure, preparation and application in auxiliary dependency recombinant adenovirus preparation thereof with helper adenovirus of hemopoietic targeting.
Background technology
Adenovirus vector is widely used in basis and the clinical research of gene therapy with advantages such as its host range wide (can infect simultaneously division cells and non-division cells), safe (unconformity enters host genome DNA), efficiency of infection is high, physicochemical property is stablized, easy preparations.Through years development, formed gradually three generations's adenovirus system:
(1) first generation recombinant adenoviral vector has been removed the E1/E3 gene expression frame, and the genes of interest about portability 7.5kb obtains recombinant adenovirus at HEK293 cell intermediate package.But the shortcomings such as it exists, and the carrier capacity is little, immunogenicity strong, the destination gene expression time is short have limited its application in gene therapy.
(2) second filial generation adenovirus vector suddenlys change to E3/E4 Gene Partial or full sequence, and the carrier capacity is expanded to 14kb, and immunogenicity obviously reduces, but the yielding poorly of ripe adenovirus particles, titre are low, and therefore, the application of second filial generation adenovirus in gene therapy is less.
(3) third-generation adenovirus vectors, be helper-dependent adenovirus vector (Helper-Dependent Adenoviral Vectors, HD-Ad), remove all encoding viral genes, only kept 5 ' and 3 ' inverted terminal repeat sequence (ITR) and packaging signal (ψ).The HD-Ad decapacitation is effectively eliminated outside the specific host immune response of virus antigen, and also having exogenous gene can be in advantages such as the medium-term and long-term stably express of cell resting stage, the significant liftings of carrier capacity.Therefore, HD-Ad has good prospect in the application of gene therapy.
The preparation of third-generation adenovirus vectors comprises three main process: make up the composition (third-generation adenovirus vectors) carry the genes of interest carrier, preparation helper virus functions such as (trans provide) virus replication packings and in package cell line intermediate package virus.At present, the packing of most HD-Ad and amplification are regulated and control by the Cre/loxp recombination system: namely insert the loxp sequence in helper virus packaging signal both sides, and preparation is viral in the package cell line of expressing the Cre enzyme.The Cre recombinase can effectively excise packaging signal, thereby suppresses packing and the amplification of helper adenovirus, improves output and the quality of purpose virus.
The serotype of HD-Ad carrier is determined by helper adenovirus, so the different serotypes helper adenovirus can be realized the serotype conversion.Based on the helper adenovirus of 5 type adenovirus vectors, its HD-Ad that is packaged to be has 5 type adenoviruss similar infection ability and targeting, and is therefore low to the efficiency of infection of hematopoietic cell, had a strong impact on its application in the hematopoietic cell gene therapy.
The transformation of adenovirus targeting can realize by transformation and the modification to fibrin, penton protein, hexon and coat protein.The process of 5 type adenovirus infection target cells at first finish by the Coxsackie-Adenovirus Receptor (Coxsackie Virus and Adenovirus Receptor, CAR) of identification cell surface, and the hematopoietic cell surface lacks the CAR receptor.For improving recombinant adenovirus to the infection ability of hematopoietic cell, the healthy and strong doctor in this laboratory Shandong etc. replace with the fiber heading part of 5 type adenovirus vectors the fiber heading sequence of B group 11p type adenovirus (Ad11p).Studies show that further the recombinant adenovirus Ad5/F 11p that changes structure can obviously strengthen the infection ability to hematopoietic cell.
In sum, take Ad5/F11p as the basis, structure and preparation have the hemopoietic targeting and are applicable to the helper adenovirus of Cre/loxp system, foundation and the application in the hematopoietic cell gene therapy thereof for the auxiliary dependency recombinant adenovirus of hemopoietic targeting prepares system have very important theory and realistic meaning.
Summary of the invention
The object of the invention is to make up and prepare a kind of helper adenovirus of hemopoietic targeting, and be applied to packing and the amplification of HD-Ad, improve HD-Ad to the infection ability of hematopoietic cell, finally provide technology platform for the application of HD-Ad in the hematopoietic cell gene therapy.
The present invention at first, transforms the packaging signal of shuttle plasmid pshuttle, and adds the loxp sequence at the packaging signal two ends take the Ad5/F11p skeleton plasmid as the basis; Secondly, with shuttle plasmid and the skeleton plasmid pAd5/F11p homologous recombination in the BJ5183 competent cell of transforming, obtain helper adenovirus plasmid pAd5/F11p-HV; Again, the helper adenovirus plasmid is after the PacI linearisation, and transfected HEK 293 is packaged to be helper adenovirus Ad5/F11p-HV; Then, carry the HD-Ad carrier pC4HSU-GFP of GFP in 293Cre4 cell intermediate package, obtain auxiliary dependency recombinant adenovirus HD-Ad5/F11p-GFP, and identify; At last, with identifying that correct HD-Ad5/F11p-GFP infects multiple hematopoietic cell, estimate it to the infection ability of hematopoietic cell.Be specially:
1, contains the synthetic of loxp site packaging signal dna fragmentation
With reference to the packaging signal sequence of Ad5 and helper virus H14, the crucial DNA sequence (highly conserved sequence) that contains in the clear and definite packaging signal is A1-A7.For reducing the packaging efficiency of helper adenovirus packaging signal, reduce helper adenovirus and pollute, improve purpose viral yield and quality.The present invention adopts incomplete packaging signal sequence A 1-A4 namely: A1:GTAAATTTG, A2:GTAAG ATTTG, A3:AGTGAAATCTG, A4:ATAATTTTG.
The loxP site is the specificity recombination site of Cre recombinase, and it is comprised of 34bp, namely
AT AACTTCGTATAGCATACAT
TATACGAAGTTAT, there is the inverted repeat (band underscore part) of 13bp at two ends, and middle 8bp determines the direction of restructuring.The loxp site is placed the packaging signal two ends, can in the package cell line of expressing the Cre enzyme, effectively excise packaging signal.
2, the transformation of shuttle plasmid pShuttle packaging signal
Above-mentioned synthetic dna fragmentation with the loxp sequence is replaced packaging signal on the pShuttle carrier, obtain recombiant plasmid pShuttle-SynES (pSh-SynEs).The shuttle plasmid that changes structure carries incomplete packaging signal, can effectively reduce packaging efficiency; And under the effect of Cre recombinase, the restructuring of loxp sequence homology can be excised packaging signal.Therefore, by the helper virus of pSh-SynEs preparation, its packaging efficiency in the 293Cre4 cell can be effectively controlled, and then reduces the content of helper virus, guarantees output and the quality of HD-Ad.
3, the structure of hemopoietic targeting helper virus, preparation and evaluation
This laboratory changes the efficient targeting hematopoietic cell of hemopoietic targeting recombinant adenovirus Ad5/F11P energy of structure, utilize shuttle plasmid pSh-SynES homologous recombination in BJ5183 of its skeleton plasmid pAd5/F11p and above-mentioned transformation, helper adenovirus plasmid pAd5/F11p-HV obtains recombinating.After the pacI linearisation, HEK293 cell packing obtains helper adenovirus Ad5/F11p-HV, and in genomic level it is identified.Identify that correct helper virus has the particle characteristics of Ad5/F11p, can the efficient targeting hematopoietic cell.
4, the Preparation and identification of hemopoietic targeting HD-Ad and contrast virus
For estimating HD-Ad to the efficiency of infection of hematopoietic cell, carry the HD-Ad plasmid pC4HSU-GFP (microbix company commercialization carrier) of GFP with helper adenovirus Ad5/F11p-HV and H14 (microbix company commercialization virus) packing respectively, respectively at preparing auxiliary dependency recombinant adenovirus HD-Ad5/F11p-GFP and HD-Ad/H14-GFP in the 293Cre4 cell.The virus for preparing is infected respectively the HepG2 cell, the expression of fluorescence microscope green fluorescent protein.The expression of correct HD-Ad visible green fluorescin.
5, the amplification of HD-Ad5/F11p-GFP and HD-Ad/H14-GFP and purification
HD-Ad5/F11p-GFP and helper adenovirus Ad5/F11p-HV infect respectively the 293Cre4 cell, and the purpose that increases in a large number virus obtains the HD-Ad5/F11p-GFP of purification through cesium chloride density gradient centrifugation.The Genome Size of HD-Ad5/F11p-GFP and Ad5/F11p-HV is respectively about 32Kb and 29Kb, and therefore, the visible HD-Ad5/F11p-GFP of caesium chloride density gradient centrifugation is positioned at the below of helper virus.
Simultaneously, HD-Ad/H14-GFP and helper adenovirus H14 amplification, purification obtain the HD-Ad/H14-GFP of purification.The Genome Size of HD-Ad/H14-GFP and H14 is respectively about 32Kb and 36Kb, and therefore, visible HD-Ad/H14-GFP is positioned at the helper virus top.
At last, detect purity and the titre of HD-Ad5/F11p-GFP and HD-Ad/H14-GFP.6, the hematopoietic cell efficiency of infection of HD-Ad5/F11p-GFP and HD-Ad/H14-GFP is estimated
After HD-Ad5/F11p-GFP and HD-Ad/H14-GFP infect Leukemia Cell Lines K562, U937, Juarket and HL-60 with different MOI, in different time points, adopt respectively its efficiency of infection of flow cytometry and fluorescence microscope, thereby estimate helper adenovirus Ad5/F11p-HV to the impact of HD-Ad hemopoietic targeting.
Innovative point of the present invention is to set up the preparation system of hemopoietic targeting HD-Ad take the hemopoietic target adenovirus as the basis.To the transformation of recombinant adenovirus packaging signal, can make it in the Cre/loxp system, under the effect of incomplete packaging signal and loxp site homologous recombination, it is residual effectively to reduce helper adenovirus, improves HD-Ad output; Simultaneously, shuttle plasmid and pAd5/F11p homologous recombination with transforming can obtain hemopoietic targeting helper adenovirus Ad5/F11p-HV.The HD-Ad that this helper adenovirus is packaged to be has the particle characteristic similar to helper virus, can the efficient targeting hematopoietic cell.
Description of drawings
Accompanying drawing 1 packaging signal dna fragmentation SynES's is synthetic: 1 is the pcr amplification product of SynES; 2 is DL2000Marker;
The evaluation of accompanying drawing 2 recombiant plasmid pSh-SynES: 1 is DL2000 Marker; 2 negative contrasts; 3 is the pcr amplification product of pMD-SynES; 4 is the pcr amplification product of pSh-SynES;
Accompanying drawing 3 helper adenovirus Ad5/F11p-HV prepare sketch map: 1 expression pSh-SynEs electricity behind the PmeI enzyme action turns the BJ5183 competent cell, with the pAd5/F11P homologous recombination, obtains pAd5/F11P-HV; The PacI linearisation of 2 expression pAd5/F11P-HV; 3 expression linearisation pAd5/F11P-HV transfected HEK 293s, packing produces helper adenovirus Ad5/F11P-HV; Accompanying drawing 4 helper adenovirus carrier pAd5/F11p-HV enzyme action are identified: 1 is λ-HindIII Marker; 2 is the EcoRV enzyme action product of pAd5/F11p-HV; 3 is the EcoRV enzyme action product of plasmid pAd5/F11p; 4 is DL2000Marker;
The evaluation a of accompanying drawing 5 helper adenovirus Ad5/F11p-HV, PCR identify: 1 pcr amplification product for the HEK293 cell genomic dna of Ad5/F11p-HV infection; 2 is the pcr amplification product of normal HEK293 cell genomic dna; 3 is DL2000Marker; B, the helper adenovirus negative staining electron microscope figure of purification;
Fluorescence microscope egfp expression a is the white light photo behind the accompanying drawing 6HD-Ad5/F11P-GFP infection U937 cell 24h; B is corresponding fluorescence photo;
48h after the HD-Ad5/F11P-GFP of accompanying drawing 720MOI and the viral HD-Ad/H14-GFP of contrast infect, Flow cytometry egfp expression result: grouping 1 is the contrast of uninfecting virus; 2 for infecting the cell of HD-Ad5/F11P-GFP; 3 for infecting the cell of HD-Ad/H14-GFP;
The specific embodiment
Its method is the synthesising packing sequence, and adds the loxp site at the sequence two ends.Behind the synthetic primer, overlapping pcr amplification obtains full length DNA sequence SynES, at last it is cloned on the pMD-18T carrier, and the evaluation of checking order.Be specially:
(1) full length DNA sequence SynES's is synthetic
Take the packaging signal sequence of Ad5 and H14 as the basis, be identified for the incomplete packaging signal sequence that helper virus makes up, add the loxp sequence at the sequence two ends, be new packaging signal DNA sequence SynES.The design primer is designated as respectively P1～P8, sees sequence table sequence 1～8, adopts overlapping pcr amplification to obtain the full length DNA sequence.
With above-mentioned primer by after waiting mole to mix, 15 circulations of first pcr amplification.Then, take above-mentioned amplified production as template, take P1 and P8 as primer, carry out second and take turns amplification, can obtain the DNA sequence SynES that total length is 241bp (shown in accompanying drawing 1a).This sequence comprises loxp site and the crucial sequence A 1～A4 of packaging signal at two ends, shown in sequence table sequence 9.
(2) pMD-SynES Vector construction
Adopt archaeal dna polymerase to add A tail rear clone to T carrier pMD-18T, Amp to synthetic SynEs sequence
+Screening obtains positive colony pMD-SynES, identifies correct through PCR, enzyme action and order-checking respectively.
The structure of embodiment 2 recombinant shuttle plasmid pSh-SynEs
Its method is with methods such as enzyme action, connections the SynEs sequence on the pMD-SynES to be transferred on the pShuttle carrier, replaces original packaging signal, obtains recombinant shuttle plasmid psh-SynEs.Be specially:
Restricted enzyme BsrGI (Bsp1407I) and BglII double digestion pMD-SynES carrier reclaim corresponding packaging signal sequence SynEs; Simultaneously, double digestion pShuttle carrier (preserving number: P3-6-1), remove packaging signal sequence Es, reclaim large fragment, be designated as pShuttle-Δ Es.Then, SynEs is connected, transforms, Kana with pShuttle-Δ Es
+Screening obtains positive colony pSh-SynEs.
The positive colony that obtains extracts plasmid through amplification, and take it as template, take P1 and P6 as primer, and the band (as shown in Figure 2) about the visible 176bp of pcr amplification.Finally, identify correctly through order-checking.
The preparation of embodiment 3 helper adenovirus Ad5/F11p-HV
Its method is as the basis take the Adeasy system, behind the PmeI enzyme action shuttle plasmid psh-SynEs, electricity turns the BJ5183-Ad5/F11p competent cell, obtains helper adenovirus plasmid pAd5/F11p-HV with Ad5/F11p skeleton plasmid homologous recombination, identifies correct through enzyme action and PCR.PAd5/F11p-HV is transfected HEK 293 preparation virus after the PacI linearisation.Genomic level identify correct after, increase, purification and titer determination.(as shown in Figure 3)
(1) generation of restructuring helper adenovirus plasmid pAd5/F11p-HV:
Skeleton plasmid Ad5/F11p (preserving number: P3-6-5) transform BJ5183 competence, Amp
+Screening obtains positive colony, after evaluation is correct, presses molecular cloning " preparation of electric shock transformed competence colibacillus cell " method, preparation BJ5183-Ad5/F11p competent cell.Shuttle plasmid pSh-SynES carries out PmeI linearisation and dephosphorylation, with the condition electric shock conversion BJ5183-Ad5/F11p competent cell of " 200 Ω, 2.5KV, 25 μ F ", Kana
+Screening obtains helper adenovirus plasmid pAd5/F11p-HV.As primer, PCR identifies correct with P1 and P6; Simultaneously, PacI, EcoRV carry out respectively enzyme action evaluation correct (as shown in Figure 4).
(2) preparation of helper adenovirus Ad5/F11p-HV:
Identify correct helper adenovirus plasmid pAd/F11p-HV behind PacI enzyme action and phenol chloroform extracting and purifying, liposome-mediated mode transfection incasing cells HEK293 (preserving number: C12-7-14).Simple microscope observation of cell form changes, 8 days visible plaque formations, and enlarge gradually, until complete pathological changes appears in 11d.Collecting cell and supernatant are collected supernatant after repeatedly freezing molten 3 times, obtain the virus stock solution used of helper adenovirus Ad5/F11p-HV, and the virus genom DNA sequence is seen shown in the sequence table sequence 10.
(3) evaluation of helper adenovirus Ad5/F11p-HV, amplification, purification and titer determination:
The virus supernatant infects the HEK293 cell, and cytopathy appears in 48～72h.Collecting cell extracts virus genom DNA, and identifies correct (shown in accompanying drawing 5a) through PCR.After identifying a large amount of amplifications of correct Ad5/F11p-HV, collect sick cell, multigelation three times is got supernatant.Behind the cesium chloride density gradient method purification, for subsequent use in Hank ' the s liquid that-80 ℃ are stored in 10% glycerol.The virus of purification is observed it and is had typical adenovirus form result (shown in accompanying drawing 5b) behind negative staining electron microscope.
Ultraviolet spectrophotometer method (wavelength 260nm) and limiting dilution assay (Median TissueCulture Infective Dose, TCID50) measure respectively granule titre (the Viral Particle Units per Milliliter of helper adenovirus Ad5/F11p-HV, vp/ml), purity (OD260nm/280nm) and infection titer (Infection Units per Milliliter, IU/ml).The result is as shown in table 1, and its infection titer, purity all meet the further experiment requirement.
The purity of table 1 helper adenovirus Ad5/F11p-HV and titer determination result
HD-Ad carrier pC4HSU-GFP is microbix company commercialization carrier, can under the assistance of helper adenovirus H14, obtain auxiliary dependency recombinant adenovirus HD-Ad/H14-GFP in 293Cre4 cell intermediate package.With reference to its preparation scheme, replace H14 with helper adenovirus Ad5/F11p-HV and prepare HD-Ad/F11p-GFP.Concrete scheme is:
(1) linearisation of HD-Ad carrier pC4HSU-GFP and transfection
The linearisation of HD-Ad carrier: the pC4HSU-GFP plasmid (preserving number: P4-3-2) carry out the PmeI enzyme action by 5 μ g/25 μ l systems, confirm can be directly used in transfection behind its Total Linearization by agarose gel electrophoresis;
Incasing cells is prepared: the 293Cre4 cell of exponential phase (preserving number: C11-6-11) with 5 * 10
5Cell/ware is laid in the 6cm culture dish;
Cell transfecting: (24～48h) time, with the mode transfection 293Cre4 cell of above-mentioned enzyme action product with the calcium phosphate mediation, and 8～12h is replaced by fresh complete medium after transfection when Growth of Cells to 30%～50% degrees of fusion.
(2) preparation of HD-Ad5/F11p-GFP
24h after the pC4HSU-GFP transfection, helper adenovirus Ad5/F11p-HV infects the 293Cre4 cell with the infection intensity of 5MOI.The fluorescence microscope green fluorescent protein produces and cell state, 48h after the transfection, and visible 90% above cell visible green fluorescent protein expression, and with a small amount of cytopathy; 72h after the transfection, visible cell are complete pathological changes state.At this moment, collect supernatant behind collecting cell and the supernatant, multigelation three times, namely obtain HD-Ad5/F11p-GFP virus stock solution used (P1).
Adopt identical strategy, adopt helper virus H14 to pack and obtain contrast adenovirus HD-Ad/H14-GFP (P1).
(3) amplification of HD-Ad5/F11p-GFP
The 293Cre4 cell of exponential phase is laid on the 15cm culture dish, when treating Growth of Cells to 80%～90% degrees of fusion, an amount of HD-Ad5/F11P-GFP virus liquid and 1MOI helper virus Ad5/F11p-HV is infected the 293Cre4 cell simultaneously.48h～72h after the transfection, the great expression of visible green fluorescin and cytopathy.At this moment, collect supernatant behind collecting cell and the supernatant, multigelation three times, namely obtain HD-Ad5/F11p-GFP.Be expanded to successively for the 6th generation, abandon supernatant, PBS collection sick cell and multigelation three times, the HD-Ad5/F11p-GFP supernatant that obtains can be used for cesium chloride density gradient centrifugation.
Adopt identical strategy, a large amount of amplifications obtain to be used for the contrast adenovirus HD-Ad/H14-GFP of cesium chloride density gradient centrifugation purification.
The purification of embodiment 5HD-Ad5/F11p-GFP and HD-Ad/H14-GFP, evaluation, purity and titre detect
HD-Ad5/F11p-GFP and HD-Ad/H14-GFP that amplification obtains, the HD-Ad that obtains purification behind cesium chloride density gradient centrifugation is viral, after enzyme action is identified correctly, measures respectively its granule titre, purity and infection titer (TCID50 method).
Concrete scheme is:
(1) purification of HD-Ad5/F11p-GFP and HD-Ad/H14-GFP
The HD-Ad5/F11P-GFP that collection obtains and HD-Ad/H14-GFP, multigelation is three times between-80 ℃ and 37 ℃, the centrifugal collection supernatant of 4 ℃ * 3500rpm * 30min.Cesium chloride density gradient centrifugation is obvious two bands as seen, are judged the position of purpose band by the Genome Size of helper adenovirus and HD-Ad.Because the genome of Ad5/F11p-HV is less than HD-Ad5/F11p-GFP, therefore, lower floor is purpose band HD-Ad5/F11p-GFP; And the genome of helper adenovirus H14 is greater than HD-Ad/H14-GFP, and the upper strata is purpose band HD-Ad/H14-GFP.After collecting the purpose band ,-80 ℃ are stored in Hank ' the s liquid that contains 10% glycerol.
(2) evaluation of HD-Ad5/F11p-GFP and HD-Ad/H14-GFP purified product
HD-Ad5/F11p-GFP and the viral HD-Ad/H14-GFP of contrast infect respectively the cells such as HepG2, PC3M, all expression of visible a large amount of green fluorescent proteins.The result shows that we have successfully prepared auxiliary dependency recombinant adenovirus HD-Ad5/F11p-GFP and HD-Ad/H14-GFP.For the further genome to HD-Ad5/F11p-GFP is identified, adopt the method for protease K digesting, the extracting of phenol chloroform obtains virus genom DNA, respectively it is carried out XhoI, the single endonuclease digestion of ScaI and KpnI identifies that the agarose gel electrophoresis testing result conforms to theoretical value.
(3) HD-Ad5/F11P-GFP and HD-Ad/H14-GFP purity and titer determination
At first, adopt ultraviolet spectrophotometry to detect respectively OD260nm, the OD260nm/OD280nm value of HD-Ad5/F11P-GFP and HD-Ad5/H14-GFP, and calculate purity and the granule titre of virus according to respective formula.
Then, adopt the TCID50 method to measure respectively the Viral infection titre.Concrete grammar is: with virus according to 10 times of doubling dilutions after, infect respectively 293 cells in 96 orifice plates, each gradient is 10 multiple holes; 48h observes each hole Green fluorescent protein expression, and selecting every hole green fluorescent protein is that 1～10 viral gradient is counted, and calculates virus titer according to following formula.
Infection titer (IU/ml)=GFP positive cell number * extension rate/virus liquid volume
Testing result is as shown in table 2, and infection titer and purity all meet further functional experiment demand, shows that successful purification has obtained HD-Ad.
Purity and the titer determination result of table 2HD-Ad5/F11P-GFP and HD-Ad/H14-GFP
Embodiment 7HD-Ad5/F11p-GFP detects hematopoietic cell targeting efficiency of infection
For estimating HD-Ad5/F11p-GFP to the targeting efficiency of infection of hematopoietic cell, take HD-Ad/H14-GFP as contrast, it is K562, U937, HL60 and Juarket cell that the HD-Ad that different MOI infect infects hematopoietic cell, in different time points, adopt respectively the expression efficiency of fluorescence microscope and Flow cytometry green fluorescent protein.Be specially:
Respectively with human leukemia cell line K562, U937, HL60 and Juarket cell with 2 * 10
5Cells/well is inoculated 24 orifice plates, and the HD-Ad5/F11p-GFP of 20MOI and 2MOI and the viral HD-Ad/H14-GFP infection cell of contrast behind 2～4h replace with culture medium fresh 1640 culture medium that contain 10%FBS.
The fluorescence microscope result shows, 24h and 48h after infecting compare with contrast virus, and HD-Ad5/F11p-GFP obviously improves the efficiency of infection of each cell line.For the U937 cell, 24h after infecting, the HD-Ad/H14-GFP infected group does not have egfp expression substantially; And the HD-Ad5/F11p-GFP infected group has the expression (as shown in Figure 6) of a large amount of green fluorescent proteins.
48h collecting cell after infecting, the expression of Flow cytometry green fluorescent protein, the result shows: viral HD-Ad/H14-GFP compares with contrast, the HD-Ad5/F11p-GFP of 20MOI all obviously improves the efficiency of infection of U937, Juarket, HL60, and has statistical significance (P＜0.01); And to the efficiency of infection of K562 cell quite (shown in table 3 and accompanying drawing 7).
The expression efficiency (%) of 48h green fluorescent protein behind the table 3 flow cytometer detection viral infection
*, compare p＜0.01 with corresponding HD-Ad/H14-GFP
In sum, we have successfully made up the helper adenovirus Ad5/F11p-HV with hemopoietic targeting, and confirm that it can be used in the preparation of HD-Ad, and the HD-Ad of its generation has the particle characteristics of helper virus, can the efficient targeting hematopoietic cell.
At first, by the transformation to the packaging signal two ends, introduced the loxp sequence, can be under the effect of Cre recombinase homologous recombination, excision packaging signal, and then reduce the pollution of helper virus guarantees the Quality and yield of purpose virus; Secondly, this helper virus is used for the preparation of HD-Ad, successfully prepares, amplification, purification obtain purpose virus, its purity and titre all meet requirement of experiment; At last, by infecting hematopoietic cell system, clear and definite its efficiency of infection to U937, Juarket and H160 is all viral apparently higher than contrast.
The coat protein of HD-Ad provides by helper virus, and therefore, the infection characterization of helper virus often plays decisive action.The helper virus of the present invention's preparation is not only applicable to the auxiliary preparation that relies on recombinant adenovirus, also can be used for the relevant hybrid virus preparation of adenovirus, and then brings into play the effect of its hemopoietic targeting.
Claims (7)
1. the restructuring helper adenovirus of a hematopoietic cell targeting, it is characterized in that take the Adeasy carrier system of stratagene company as the basis, fiber heading sequence with B group 11p type adenovirus (Ad11p) is replaced the partial sequence that 5 type adenovirus fibers are headed a ball, and namely obtains recombinant adenovirus Ad5/F11p; Incomplete packaging signal sequence A 1～A4 with 5 type adenoviruss replaces original packaging sequence, and adds the loxp sequence at its two ends, and the loxp sequence at described incomplete packaging signal sequence A 1～A4 and two ends thereof is sequence shown in the sequence 9; Finally obtain the helper adenovirus Ad5/F11p-HV that recombinates.
2. restructuring helper adenovirus according to claim 1 is characterized in that it has the hematopoietic cell targeting.
3. restructuring helper adenovirus according to claim 1 is characterized in that its 1oxp sequence that contains can effectively excise packaging signal in the 293Cre4 cell, reduces helper virus output.
4. restructuring helper adenovirus according to claim 1 is characterized in that it can be at the auxiliary dependency recombinant adenovirus of 293Cre4 cell intermediate package.
5. restructuring helper adenovirus according to claim 4, wherein said auxiliary dependency recombinant adenovirus has the hematopoietic cell targeting.
6. the application of each described restructuring helper adenovirus of claim 1-5 in the auxiliary dependency recombinant adenovirus of preparation.
7. the auxiliary dependency recombinant adenovirus for preparing of each described restructuring helper adenovirus is for the preparation of the application in the medicine of hematopoietic cell target gene therapy according to claim 1-5.
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