CN100588709C - Defected slow virus vector derived from HIV-1 virus - Google Patents
Defected slow virus vector derived from HIV-1 virus Download PDFInfo
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- CN100588709C CN100588709C CN200610038217A CN200610038217A CN100588709C CN 100588709 C CN100588709 C CN 100588709C CN 200610038217 A CN200610038217 A CN 200610038217A CN 200610038217 A CN200610038217 A CN 200610038217A CN 100588709 C CN100588709 C CN 100588709C
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Abstract
The invention discloses a defective slow virus carrier form HIV-1 virus, which comprises the following steps: using cis-form action element of HIV-1 genom and slow virus carrier pLXB from sequence ofreaction type protein as initial carrier; obtaining pLXB-Sin by linking up with initial carrier and pHit-Sin carrier of removed U3 district 3'-LTR fragment; inserting WPRE element and EGFP; obtainingpLenti-GFP. The invention possesses infected non-cleavage cell, cleavage cell and goal DNA sequence.
Description
Technical field:
The present invention relates to novel lentiviral vectors, especially a kind of defective type lentiviral vectors that comes from HIV-1 virus.
Background technology:
The non-virus carrier transfection efficiency is lower, and the gene therapy scheme great majority that are used for human experimentation carry out transgenosis with virological method, and is wherein ripe with reverse transcription coe virus and adenovirus carrier.Adenovirus carrier is obtained very big achievement in gene therapy, its transfection efficiency height, enough big host range is arranged, can infect resting cell, but the goal gene unconformability is to the target cell genome, only can transient expression, and some antigenicity of adenovirus itself can cause human immunity reaction, stops it to repeat transduction.
Modern medicine study proves that a lot of human difficult diseases of people all have directly with gene gets in touch.High and the high disease of mortality ratio for sickness rate such as cancers, the treatment plan from gene is started with and designed can reach the effect that has no side effect.
In gene therapy, can use gene engineering, gene is imported in the cell, needn't the medication disease just can cure.Although gene therapy has obtained than much progress, there are some problems to need to be resolved hurrily, such as the introducing carrier of gene and method, the screening of target gene, the organ target of gene therapy.The solution of these problems will bring breakthrough progress for gene therapy, also bring brand-new hope for the patient.Especially the carrier introduced of gene and the method key that is whole technique, can goal gene effectively change recipient cell over to, depends on used carrier to a great extent.
Lentivirus is in Retroviridae, but its genome structure complexity, except that these 3 structure genes similar with simple retrovirus of gag, pol and env, also comprises 4 auxiliary genes, vif, vpr, nef, vpu and 2 regulatory gene tat and rev.HIV-1 is the distinctive virus of tool in the slow virus, and first slow virus carrier system is that the basis makes up with this virus promptly.
The structure principle of first-generation lentiviral vectors is exactly that the cis-acting elements in the HIV-1 genome (as packaging signal, long terminal repeat) is separated with the proteic sequence of coding trans-acting.Carrier system comprises packing composition and carrier components: the packing composition has been removed the required cis acting sequence of packing, reverse transcription and integration by the HIV-1 genome and has been made up, and can transly provide to produce the required albumen of virion; Carrier components and the complementation of packing composition contain the required HIV-1 cis acting sequence of packing, reverse transcription and integration.Have the multiple clone site under the allogeneic promoter control simultaneously and insert people's goal gene in this site.For reducing the possibility that two kinds of composition homologous recombination produce the virus (RCV) that replication is arranged, change the 5 ' LTR that packs composition into cytomegalovirus (CMV) immediate early promoter, 3 ' LTR changes SV40 polyA site etc. into.To pack composition and be structured in respectively on two plasmids, promptly one express gag and pol, another expresses env.Three plasmid expression systems have been made up according to above principle.Three plasmid expression systems comprise packaging plasmid, envelope protein plasmid and transferring plasmid.Wherein packaging plasmid is expressed HIV 1 and is duplicated required whole trans-activators, but do not produce virus envelope protein and accessory protein vpu under the control of CMV promotor; The plasmid-encoded vesicular stomatitis virus G of envelope protein albumen (VSV-G), the false configuration lentiviral vectors of using the VSV-G coating has enlarged the target cell preferendum scope of carrier, and increased the stability of carrier, and allow carrier to be concentrated by high speed centrifugation, improved titre.1., virus titer is low however, first-generation lentiviral vectors still has following significant disadvantages:; 2., the host range that can infect is narrow; 3. the chance that produces RCL is higher relatively, and biological safety is relatively poor.
S-generation lentiviral vectors is to have removed the composition Vif and the Nef that may make the dormant composition Vpr of target cell in the first-generation carrier, can suppress target cell growth even long-living apoptosis, thereby makes that this carrier is safer.But still there is safety issue in s-generation lentiviral vectors.
In order to satisfy the needs of gene therapy, wish that the virus vector of a new generation meets following feature:
1. high transfection, can effectively infect division stage cell and not division stage cell: carrier should be in vivo efficient various tumour cells of transfection.Although in the past therapy of tumor, people thought once that only infecting the division stage cell carrier can target import tumour cell, but most researchers thinks that tumour not only has the division stage of being in cell now, also have and be in not the division stage cell, singly kill and be in the division stage tumour cell, be not enough to kill all tumour cells.Therefore carrier should efficiently infect division stage and not division stage tumour cell.
2. tumour cell target sexuality is dyed: carrier can only the specific infection tumour cell, and does not influence normal cell.
3. tumour-specific and controllable express: goal gene efficiently expresses at specifically inside tumor cell, and does not express in normal cell.
4. weak immunogenicity: should not cause immune response after carrier changes over to.
5. produce easily: carrier should be produced and reach commercial-scale high titre product.This comes from, and we will import a large amount of cells usually, if widespread use, carrier also will adapt to the needs of commercial production, manufacturing, and transportation has certain preservation period easily.
Summary of the invention
The objective of the invention is to: the virus titer that exists at first-generation lentiviral vectors is low, and the host range that can infect chance narrow and generation RCL is higher relatively, and biological safety is relatively poor; S-generation lentiviral vectors does not still solve the practical problems of security, and a kind of new defective type lentiviral vectors that comes from the HIV-1 C-type virus C is provided.
The object of the present invention is achieved like this: a kind of defective type lentiviral vectors that comes from HIV-1 virus, it is characterized in that, with the cis-acting elements in the HIV-1 genome (as packaging signal, long terminal repeat) and the isolating lentiviral vectors pLXB of the proteic sequence of trans-acting as initial carrier, with its with remove U3 zone 3 '-the segmental pHit-Sin carrier of LTR is connected and obtains pLXB-Sin, inserts WPRE (post-transcriptional regulatory element from the genome ofWoodchuck hepatitis virus) element again, EGFP (enhanced GFP) obtains pLenti-GFP.
In the present invention, with lentiviral vectors pLXB as initial carrier, cut with Xba I and Pst I enzyme, reclaim big fragment, with identical double digestion digestion pHit-Sin carrier, obtain to have removed 3 of U3 zone '-the LTR fragment, connect acquisition pLXB-Sin with the T4DNA ligase enzyme, then pLXB-Sin is cut with Pst I and BamH I enzyme, reclaim big fragment; The WPRE fragment is downcut from pSK-WPRE with Pst I and Sal I enzyme; With EGFP? downcut from pEGFP-Cl with Sal I and BamH I, then above three fragments are made three fragments with the T4DNA ligase enzyme and connect, obtain pLenti-GFP.
In the present invention, when changing cell over to, from packaging plasmid, removed the Tat element.
The invention has the advantages that:, make that this carrier is safer owing to removed the wild-type virus element that may cause replicative lentivirus to produce in the packaging plasmid.Removed the gene order of the terminal U3 of 3 '-LTR zone 133-bp simultaneously, thereby make it to become self disactivation carrier (SIN carrier), help producing the carrier with tissue-specific promoter, when being used for the SiRNA gene therapy, U6 or H1-RNA promotor can not be subjected to the influence of 5 '-LTR.Because of having lost 5 '-LTR strong promoter function, obviously reduced the danger that causes canceration because of the insertion inactivation simultaneously.Have can infect Unseparated Cell and somatoblast, can insert big segmental target dna sequence, can not cause insert inactivation, can make foreign gene in target cell stably express, be difficult for bringing out advantages such as host immune response, biological safety are good.
Description of drawings:
Fig. 1 is the structural representation of pLenti-GFP;
Fig. 2 is the non-fluorescence photo of Hela-pLenti-GFP;
Fig. 3 is the fluorescence photo of Hela-pLenti-GFP.
Specific implementation method:
The structure of embodiment 1, pLenti-GFP.
(1), cis-acting elements (as packaging signal, long terminal repeat) in the use HIV-1 genome and the isolating lentiviral vectors pLXB of the proteic sequence of trans-acting are as initial carrier, it is cut with Xba I and Pst I enzyme, reclaim big fragment, with identical double digestion digestion pHit-Sin carrier, obtain to have removed 3 of U3 zone '-the LTR fragment, connect acquisition pLXB-Sin with the T4DNA ligase enzyme.
(2), pLXB-Sin is cut with Pst I and BamH I enzyme, reclaim big fragment; The WPRE fragment is downcut from pSK-WPRE with Pst I and Sal I enzyme, EGFP is downcut from pEGFP-C1 with Sal I and BamH I, then above three fragments are made three fragments with the T4DNA ligase enzyme and connect, obtain pLenti-GFP (structure such as Fig. 1), enzyme is cut and is identified that it inserts correctly.
The production of embodiment 2, pLenti-GFP slow virus supernatant.
1., inoculation 10 in 100mm culture dish (Corning)
5Individual 293T cell is treated to do when cell grows to 70-80% and merges to go down to posterity at 1: 3, as substratum, cultivates 24h with the DMEM (purchasing the company in GIBCO-BRL) that contains 10% calf serum (purchasing in Hangzhou folium ilicis chinensis company) in the 5%CO2 incubator.
2., when treating that cell reaches 70% fusion, pLenti-GFP plasmid lentiviral vectors with the packaging plasmid of having removed the Tat element, is carried out transfection with the coprecipitation of calcium phosphate method.
3., treat to change cell culture medium behind the cell growth 24h, put in 37 ℃ of 5%CO2 incubators and cultivate, the viral supernatant of results changes cell culture medium behind the 24h.
4., results transform back 72h virus supernatant, handle Tissue Culture Dish with SYNTHETIC OPTICAL WHITNER and produce cell with break virus.
5., viral supernatant is made the 50000rpm ultracentrifugation, draw viral supernatant layer.
6., survey virus titer, adjusting virus titer with PBS is 1012VP/ml.
7., use the filter membrane of 0.22 μ m to carry out ultrafiltration, be packaged into then in the cillin bottle of 2ml (every cillin bottle contains 1ml).-80 ℃ profound hypothermia refrigerator is preserved.
8., virus carried out thermal source detects, microorganism detection, whether have wild virus to duplicate (RCL) with the detection of PCR method simultaneously, guaranteeing does not have thermal source, microbial contaminations such as no bacterium, fungi, virus, no wild virus duplicates.
The evaluation of embodiment 3 transfection efficiencies
Inoculation 10 in 100mm culture dish (Corning)
6Individual Hela cell is put among DMEM substratum (purchasing the company in the GIBCO-BRL) 10ml that contains 10% foetal calf serum (available from the Hangzhou folium ilicis chinensis) and is cultivated, and goes down to posterity by 1: 3 when treating cell 80% fusion.
Inhale behind the 24h and remove substratum, add supernatant 5ml, put in 37 ℃ of incubators and replenish fresh DMEM substratum 5ml behind the cultivation 3h; Do the pLenti-GFP slow virus infection once with method behind the 24h, go down to posterity behind the cultivation 24h in 37 ℃ of incubators, take the photograph photo with fluorescent microscope.Fluorescence photo and Fig. 2 compare among Fig. 3, after being presented at lentiviral vectors and infecting almost 100% cell be green cell, efficiency of infection nearly 100%.
Claims (2)
1, a kind of defective type lentiviral vectors that comes from HIV-1 virus, it is characterized in that, with the cis-acting elements in the HIV-1 genome and the isolating lentiviral vectors pLXB of the proteic sequence of trans-acting as initial carrier, with its with remove U3 zone 3 '-the segmental pHit-Sin carrier of LTR is connected and obtains pLXB-Sin, inserts the WPRE fragment again, EGFP obtains pLenti-GFP;
PLenti-GFP makes up like this: with lentiviral vectors pLXB as initial carrier, cut with Xba I and Pst I enzyme, reclaim big fragment, with identical double digestion digestion pHit-Sin carrier, obtain to have removed 3 of U3 zone '-the LTR fragment, connect acquisition pLXB-Sin with the T4DNA ligase enzyme, then pLXB-Sin is cut with Pst I and BamH I enzyme, reclaim big fragment, obtain first fragment; The WPRE fragment is downcut from pSK-WPRE with Pst I and Sal I enzyme, as second fragment; EGFP is downcut from pEGFP-C1 with Sal I and BamH I, as the 3rd fragment; With the T4DNA ligase enzyme above first, second, third fragment is connected then, obtain pLenti-GFP.
2, the defective type lentiviral vectors that comes from the HIV-1 C-type virus C according to claim 1 is characterized in that: the Tat element in the packaging plasmid should be removed when changing cell over to.
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| CN200610038217A CN100588709C (en) | 2006-02-10 | 2006-02-10 | Defected slow virus vector derived from HIV-1 virus |
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| CN200610038217A CN100588709C (en) | 2006-02-10 | 2006-02-10 | Defected slow virus vector derived from HIV-1 virus |
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| CN100588709C true CN100588709C (en) | 2010-02-10 |
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| CN110699381A (en) * | 2019-09-17 | 2020-01-17 | 合肥瑞灵生物科技有限公司 | Mediterranean anemia gene therapy vector construction method and application thereof |
| CN112760342B (en) * | 2019-11-05 | 2023-03-28 | 珠海联邦制药股份有限公司 | Lentiviral vector and cell strain for activity determination of GLP-1 and analogue thereof |
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Non-Patent Citations (4)
| Title |
|---|
| HIV-1慢病毒载体的构建及结构改造. 李振宇等.中华血液学杂志，第25卷第9期. 2004 |
| HIV-1慢病毒载体的构建及结构改造. 李振宇等.中华血液学杂志，第25卷第9期. 2004 * |
| Lentiviral vectors for enhanced gene expression in humanhematopoietic cells. Ali Ramezani et al.molecular therapy,Vol.2 No.5. 2000 |
| Lentiviral vectors for enhanced gene expression in humanhematopoietic cells. Ali Ramezani et al.molecular therapy,Vol.2 No.5. 2000 * |
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