CN104673835A - Efficient mediated Lnc-MEG3 gene overexpression lentiviral vector - Google Patents
Efficient mediated Lnc-MEG3 gene overexpression lentiviral vector Download PDFInfo
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Abstract
The invention mainly relates to an lentiviral vector system of efficient mediated Lnc-MEG3 gene overexpression in the mammal, particularly in the hematopoietic cell. The vector is constructed based on the pCDH-EF1-MCS-T2A-copGFP and the packaging plasmid pc DNA3.0-LNC-MEG3, so that high virus titer and expression quantity can be generated and the lentiviral vector system can be applied in the further tumour research and related disease treatment.
Description
Technical field
The present invention relates to biotechnology and field of gene, be specially a kind of foundation of slow virus carrier system of efficient mediation lnc-MEG3 gene overexpression.
Background technology
LncRNA (long non-coding RNA, lncRNA) is the non-coding RNA that a class transcript length is greater than 200bp, can be used as the regulatory molecule that in human genome, a class is important and plays its biological function in several ways.Research in recent years shows, lncRNA also can as the competitive endogenous RNA of one (competing endogenous RNA, ceRNA) interacts with 10miRNA, the expression regulation of participation target gene, and play an important role in the developing of tumour.
LncRNAs is as a kind of novel tumor markers, and it plays very important effect in the regulation process of tumorigenesis, and this mechanism can treat for people the thinking that tumour provides new.Current people know little to the biology of most lncRNAs and molecular characterization, but along with information biology development, secondary structure and tertiary structure analyses can provide important means for the research of lncRNAs, interaction relationship particularly between itself and encoding sequence, the development in this direction will expand out brand-new research field.Shown by the result of clinical observation and experimental study gained in a large number, the unconventionality expression of lncRNAs can cause the generation of a lot of disease, and this mechanism is particularly important in cancer biology.Although the mechanism of action relating to most of lncRNAs of tumour is not also accurately illustrated, but find with regard to its research to the known lncRNA of part, it participates in the regulation and control of chromatin reconstruct and histone modification, particularly regulates and controls by the modification of epigenetic generation and the migration that (such as gene imprinting and histone modification) can affect tumour.Such as, HOTAIR by regulation and control chromatin site reset promote metastases, and linc P21 by p53 approach, by RNA, " DBP hnRNPK is attached on chromatin and realizes its inhibitor effect.
Maternal expressing gene 3 (MEG3) is the maternal expressing gene of mouse found in 2000 by Miyoshi etc. first, it is gene trap locus 2 (gene traplocus2, Gtl2) human homolog, be positioned karyomit(e) 14q32.3, length is about 1.6kb.MEG3 can encode out a lncRN A at many normal tissue expressions, but it is disappearance (comprise genetically deficient, between promotor high methylation and gene, the mechanism and multiple such as to methylate in site is the principal element causing MEG3 genetically deficient to express) in primary tumor patient and tumor cell line.Research finds, the expression again of ME G3 can promote apoptosis, thus inhibition tumor cell propagation.M EG3 can also cause the accumulation of p53, stimulates the transcriptional activation of p53 mediation and optionally regulates and controls p53 expression of target gene.Such as in mouse, the removal of MEG3 can cause mice skeletal defect and perinatal death, and the inactivation of MEG3 can cause Angiogensis genetic expression increase and microvascular in brain to be formed, these evidences all show that MEG3 can as a lncRNA tumor inhibitor.
At present the research of LncRNA is just risen in this world, and LNC-MEG3 is imprinted gene, it is high expression level in the normal tissue, and low expression in tumor tissues, prove that it has the function of cancer suppressor gene at present, so, how to improve its expression efficiency in tumour cell and then the functional gene of its cancer suppressor gene identified, needing the instrument of efficient, stable process LAN gene studies.
Efficient gene treatment depends on exogenous gene high-efficient, stable expression.The method of tumor antigen gene transfered cell generally has two large classes: one is the virus vector method that is carrier with adenovirus, retrovirus, poxvirus and adeno-associated virus (AAV) etc.; Two is the non-virus carrier methods such as DNA liposome complex, calcium phosphate precipitation, electroporation.The advantage of non-virus carrier method transfection is: Insert Fragment size is unrestricted; Immunogenicity is low; Produce easy.Its shortcoming is that the transfection efficiency of foreign gene is general very low.Virus vector mediated method, becomes most widely used method in therapy of tumor with its high transfection efficiency and good targeting.Utilize the vaccine that the encoding gene loaded dendritic cells with tumour or virus antigen is prepared by virus vector, due to specific antigens corresponding to continuous expression can be processed in cell, the antitumor or Immune responses of the antivirus of inducing producing specificity and receiving much concern.Wherein the most frequently usedly comprise the carriers such as retrovirus (retrovirus), adenovirus (adenovirus) and adeno-associated virus (adeno-associated virus, AAV).
Slow virus (Lentivirus) carrier is the gene therapy vector grown up based on HIV-1 (human immune deficiency I C-type virus C).Distinguish general retroviral vector, it all has infection ability to somatoblast and Unseparated Cell.
Conventional virus vector has adenovirus, retrovirus and slow virus.Retroviral vector can only infect division cells, and finite capacity, and adenovirus generally can not be incorporated on karyomit(e), can only carry out transient infections.Compared with other retrovirus, slow virus (LV) has can infect non-division cells, it is large to hold allogenic gene fragment, can the remarkable advantage such as long-term expression.Slow virus does not produce any effective cellullar immunologic response, can be used as the instrument of a kind of outer-gene transport.The transgene expression energy periods of months of lentivirus-mediated, and without observable pathological phenomena.
Slow virus packaging system is generally made up of Lentiviral and slow virus package carrier.
And slow virus packaging plasmid can provide all transcribing and pack all accessory proteins required for RNA to the Pseudovirion vector of recombinating.For producing the virion of high titre, need to utilize expression vector and packaging plasmid cotransfection cell simultaneously, in cell, carry out the packaging of virus, packaged pseudovirion is secreted in extracellular substratum, after centrifuging and taking obtains supernatant liquor, the infection of host cell can be directly used in.
In prior art, using retrovirus, adenovirus to carry lnc-MEG3 gene, to carry out the effect of cell transfecting unsatisfactory, there is expression amount virus titer inadequate, the defects such as recombinant expressed amount is on the low side.And in lentiviral vectors, also not all kinds is all used in and carries lnc-MEG3 gene.
Based on such demand, this invention exploits the Lentiviral that stability and high efficiency expresses LNC-MEG3 goal gene.
Summary of the invention
The object of the invention is to build a kind of slow virus that efficiently can mediate the transgenosis of lnc-MEG3 process LAN, for providing a kind of strategy newly based on the targeted therapy of lnc-MEG3.
The present invention is with slow virus carrier system pCDH-EF1-MCS-T2A-copGFP, and based on packaging plasmid pcDNA3.0-LNC-MEG3, the slow virus of finally preparing of being recombinated by goal gene LNC-MEG3 and plasmid pCDH-T2A-GFP containing LNC-MEG3 gene, will have the targeting of LNC-MEG3 gene.GFP is selected to observe the efficiency of infection of improved lentiviral vectors pCDH-EF1-MCS-T2A-copGFP-Lnc-MEG3 to different tumour cells such as 293 cells, the primary CD34+ cell of marrow, KCL22 cell, XG7 cell, SKO-007 cells as reporter gene, detect the efficiency of metainfective Lnc-MEG3 process LAN respectively, also observe the slow virus after process LAN Lnc-MEG3 gene to the impact of cellular biology of tumor characteristic simultaneously.
Vector construction
The Lentiviral selected in the application is pCDH-EF1-MCS-T2A-copGFP (7.26Kb) and (commercially available in www.systembio.com, Cat.#CD521A-1), this carrier is the lentiviral vectors based on HIV, go for polytype division and non-division host cell, by EF1a promoters driven goal gene be transcribed into bicistronic mRNA with resistant gene; Therebetween containing one section of T2A peptide, can self splicing in level upon translation, ensure that goal gene is identical with resistant gene translation skill.GFP gene integration is entered this lentiviral vectors, forms pCDH-EF1-MCS-T2A-copGFP, make carrier can express can the green fluorescent protein that detects by flow cytometer.
Goal gene
The goal gene selected in the application is lnc-MEG3, this goal gene is provided by carrier pcDNA3.0-LNC-MEG3, wherein carrier pcDNA3.0 is the carrier that Molecular Cloning: A Laboratory room routine uses, according to the gene order design and synthesis special primer of MEG3 in GenBank, increase the cDNA sequence of MEG3 from liver cancer cell, be built in pcDNA3.0 carrier for expression of eukaryon, building process is see accompanying drawing 2.The carrier carrying goal gene obtained is for the structure of recombinant slow virus expression vector of the present invention.
Beneficial effect
Attempt in the experimentation of the application employing multiple expression vector, result shows, the effect of Lentiviral is better than retrovirus and adenovirus, and best virus titer can be obtained with the recombinant vectors constructed by pCDH-EF1-MCS-T2A-Puro lentiviral vectors in Lentiviral, transformation efficiency can up to 80%, and the expression amount of goal gene can improve 300 times relative to the cell for transforming, and can improve more than 2 times relative to other lentiviral vectorss.
Detection method
The detection method related in the application comprises agarose gel electrophoresis, flow cytometer fluorometric analysis, bacterium colony PCR method and qPCR detection method.These methods all use this area routine techniques means to be realized.
Purposes
The suitable host of the recombinant vectors constructed by the application comprises mammalian cell, preferred human cell, more preferably hematopoietic cell, most preferably 293T cell.This carrier can be used for the effect of research lnc-MEG3 gene in tumour generating process, can be further used for prevention or the therapeutic action of studying lnc-MEG3 gene pairs kinds of tumors.
Accompanying drawing explanation
Fig. 1 pCDH-EF1-MCS-T2A-copGFP vector plasmid collection of illustrative plates and multiple clone site information
The structure of Fig. 2 slow virus over-express vector pCDH-EF1-MCS-T2A-copGFP-MEG3.
The agarose gel electrophoresis figure of Fig. 3 pcDNA3.0-MEG3 plasmid, swimming lane 1, Mark er, D2000; Swimming lane 2, MEG3 object fragment; Swimming lane 3, Marker, HindIII.
Fig. 4 clones the agarose gel electrophoresis figure of the MEG3 fragment of acquisition, swimming lane 1, Ma rker, D2000; Swimming lane 2, PCR primer, MEG3 fragment
Fig. 5 carrier and object fragment double digestion, the agarose gel electrophoresis figure being connected rear recombinant vectors, swimming lane 1, Marker, HindIII; Swimming lane 2, carrier; Swimming lane 3, M arker, D2000; Swimming lane 4, MEG3.
Fig. 6 recombined lentivirus vector connects the qualification of product, swimming lane 1, Marker, Hind III; Swimming lane 2-6, connects product; Swimming lane 7, Marker, D2000.
Fig. 7 connects product P CR and identifies agarose gel electrophoresis figure, swimming lane 1, Marker, 2000; Swimming lane 2, pcDNA3.0-MEG3; Swimming lane 3, H2O; 4-8, connects product.
Fig. 8 recombinant vectors green fluorescence transfection efficiency detects, and wherein left side is contrast, and right side is fluoroscopic examination result after recombinant vectors transfection
Destination gene expression Efficiency testing after the transfection of Fig. 9 recombinant vectors
Figure 10 flow cytometer detection efficiency of infection analysis chart
The expression change of goal gene MEG3 after Figure 11 Transfected Recombinant Plasmid 293 cell
Embodiment
It is to be noted, round pcr involved by the application is the routine techniques means related in molecule clone technology handbook, the enzyme wherein related to, primer, reagent and reaction conditions all can carry out choose reasonable according to the experience of those skilled in the art when not specified (NS), the biomaterial wherein related to all belongs to commercially available usual production when indicating, and the detection means wherein related to and instrument are also well known to the skilled person and skillfully grasp.
The structure of embodiment 1MEG3 recombined lentivirus vector
1) pcr amplification of MEG3
This is primer sequence:
MEG3V1-FXbaI:
TGCTCTAGAGCAGAGAGGGAGCGCGCCTTG
MEG3V1-RNotI:
ATTTGCGGCCGCCACATTTATTGAGAGCACAGTG
Concrete steps:
PcDNA-3.0-MEG3 (building mode is see detailed Description Of The Invention part and accompanying drawing 2) plasmid is diluted 10 times, and getting 1ul is that template carries out pcr amplification, prepares 50 μ l reaction systems as follows:
After qualification, under ultraviolet lamp, cut the target DNA band of 1.6kb.DNA fragmentation sepharose reclaims by reclaiming test kit (TIANgel Midi Purific) operation.
2) enzyme of PCR primer is cut
Double digestion is carried out to PCR primer and lentiviral vectors carrier pCDH-EF1-MCS-T2A-copGFP respectively with restriction enzyme Xba I, Not I.
PCR primer double digestion reaction system is:
Carrier double digestion reaction system is:
37 DEG C of water-bath 4h.
Digestion products carries out agarose gel electrophoresis, cuts target DNA band (1.6kb) under ultra violet lamp, carrier DNA band (7.2kb).The DNA fragmentation sepharose that enzyme is cut reclaims.
3) (being connected with carrier segments) is connected
Linked system is:
After mixing, 16 DEG C of water-baths connect spends the night.
The recombinant plasmid of acquisition is carried out agarose gel electrophoresis, the connection product of results 8.86kb fragment.
4) transform
1. competent cell (JM109/DH5 α) is placed on thawed on ice 5min.
2. 20 above-mentioned μ l are connected product and put into competent cell, mix gently.
3. 30min is placed on ice.
4. heat shock 90s, then place 5min on ice.
5. the LB liquid nutrient medium of 600 μ l antibiotic-frees is added, 37 DEG C of 180rmp shaking culture 60min.
6. the centrifugal 5min of 3000rpm, only stays about 100 μ about l liquid to be mixed by bacterium.
7. bacterium liquid is added on LB flat board, even with aseptic spreading rod coating, be inverted overnight incubation for 37 DEG C.
5) bacterium colony PCR identifies recombinant vectors
Clone after colony polymerase chain reaction (PCR) method qualification transforms.
PCR reaction system is:
Forward primer (10 μMs) people MEG3-f:5 '-GCTCTACTCCGTGGAAGCAC-3 ' 1 μ l
Reverse primer (10 μMs) people MEG3-R:5 '-CAAACCAGGAAGGAGACGAG-3 ' 1 μ l
2×Taq starmix with loading Dye 10μl
Sterilized water polishing to 20 μ l
PCR primer, by after double digestion, detects the signature band of 1.6kb with agarose gel electrophoresis.Be tested and appraised, obtain recombined lentivirus vector pCDH-EF1-MCS-T2A-copGFP-LNC-MEG3.
The transfection of embodiment 2 recombined lentivirus vector and amplification
1) cell transfecting
Inoculation 293T cell 4-4.5 × 106 are in 10cm culture dish.Standby next day treats that cell is paved with to 80-85%, carries out slow virus plasmid transfection.
Calcium phosphate transfection method by slow virus plasmid transfection to 293T cell:
1) pMD2G:3ug, pSAX2:9ug, object plasmid: 12ug.Three kinds of plasmid mixings.(plasmid is necessary for large extraction reagent kit and extracts, and strict control of quality, concrete steps are shown in QIAGEN test kit specification sheets.)
2) CaCL2 of 63ul 2.0M is added.
3) with 0.1 × TE polishing to 500ul, mixing.
4) slowly instilled by aforesaid liquid in 500ul 2 × HBS, limit edged mixes.
5) be muddy state after liquid blending, room temperature leaves standstill 25-30min.
6) 1ml mixed solution is slowly instilled in 1 culture dish.
Hour 2.2.36-7, after, check cell state, liquid in culture dish be changed to 6ml fresh medium (liquid is preheated to 37 DEG C) gently and add 36ul Sodium propanecarboxylate.
Change respectively at 24h, 36h and 48h after liquid, collect supernatant liquor and be stored in 4 DEG C temporarily, and change 6ml fresh medium+36ul Sodium propanecarboxylate.
Last by all collection centrifugals: 3500rpm, 15min, 4 DEG C, abandon precipitation.Supernatant and PEG mix with 4:1, and 4 DEG C are spent the night.
Centrifugal: 3000rpm, 30min, 4 DEG C, abandon supernatant.To precipitate resuspended to (on ice operation) in suitable volumes IMDM, carry out virus titer mensuration.-80 DEG C of preservations after virus liquid packing.
Use the same method inoculation CML-CD34+ cell KCL22 cell, K562 cell preserve virus liquid.
Inoculation HT1080 cell 2 × 105/2ml is in six orifice plates.Standby next day carries out slow virus infection.
Virus after purifying or stoste are infected in each hole HT1080 cell by 1 μ l/ hole, 5 μ l/ holes or 100 μ l/ holes, 500 μ l/ holes.Add polybrene, 4 μ l/ holes simultaneously.In the process, get a hole do not add virus cell carry out count (general cell can be bred to 4 × 105/ holes); After counting, remaining cell can be continued to put into hole and be used as blank cell.
After infecting virus-4 8h, peptic cell also carries out flow cytometer detection (GFP or RFP etc.) after washing 1 time with PBS.
Embodiment 3 slow virus titer determination
Adopt Flow Cytometry, collect the 293T cell after transfected virus and CML-CD34+ cell KCL22 cell, K562 cell, after washing, the ratio of upflowing machine testing GFP.
Can in fluorescence microscopy Microscopic observation luciferase expression situation during virus infection.
According to cell count and GFP (RFP etc.) flow cytometer detection result, calculate virus titer.
Titre (titer unit/ml)=[-ln (non-infected cells ratio) × every porocyte number]/drip virus (ml)
By observing the ratio with the 293T cell of fluorescence under stream type cell analyzer, result shows, has the cell of more than 80% to have fluorescence, shows that the transfection efficiency of recombinant vectors in cell can reach more than 80% (see Fig. 8).
Embodiment 4 destination gene expression detects
Detected by qPCR method the expression of the goal gene MEG3 after transfection, compared with expressing with the MEG3 in 293T cell before infecting, expression amount improves 300 times.
PCR primer:
People MEG3-f:5 '-GCTCTACTCCGTGGAAGCAC-3 '
People MEG3-R:5 '-CAAACCAGGAAGGAGACGAG-3 '
QPCR condition:
Adopt: real-time fluorescence quantitative PCR
Reaction mixture 20ul, containing 0.5ul cDNA, 10ul 2 × SYBR Premix Ex Taq (TaKaRa biotech company (Dalian)), 0.4ul ROX-II, every 10mM mixture 0.8ul forward and reverse primer, 7.5ul water.
PCR reaction starts 30s at 95 DEG C, before 40 thermal cyclings, and 3s at 95 DEG C, 30s at 60 DEG C at every turn.Data according to comparing the analysis of CT value method, and carry out stdn by the β expression of actin in each sample.Produce the solubility curve that each PCR reacts, to ensure the purity of amplified production.
The carrier system that the present invention builds, detect GFP efficiency through fluorescence microscope fluorescence intensity, Flow Cytometry, the multiple method such as expression of qPCR technology for detection goal gene all confirms, transfection efficiency in hematopoietic cell all reaches more than 80%, the lifting amplitude of goal gene reaches the rising of 300 times, meets the research carrying out functional experiment completely.
Embodiment 5 destination gene expression efficiency comparative
MEG3 gene clone is entered AdEasyTM transfer vector, form recombinant adenoviral vector AdEasy-MEG3 (contrast 1), MEG3 gene clone is entered MSCV retroviral vector, forms recombinant retroviral vector MSCV-MEG3 (contrast 2).Above-mentioned two kinds of recombinant vectorss incorporate GFP gene simultaneously.The recombined lentivirus vector pCDH-EF1-MCS-T2A-copGFP-LNC-MEG3 obtained by above-mentioned two kinds of carriers and the application is difference transfection 293T cell under identical sample size and experiment condition, analyze virus titer according to the drain cell analytical method described, analyze the destination gene expression situation of three kinds of recombinant vectorss according to the destination gene expression detection method described.The results are shown in following table,
Table 1
For the expression of goal gene, almost do not observe the expression of MEG3 gene when using adenovirus, and in retroviral situation, the expression efficiency of MEG3 is only 36% of the application's lentiviral vectors.Result shows, selects Lentiviral, and especially the pCDH-EF1-MCS-T2A-copGFP-LNC-MEG3 expression vector of the application can obtain the highest expression efficiency.
Claims (7)
1. a recombinant slow virus expression vector, is characterized in that building based on pCDH-EF1-MC S-T2A-copGFP, and is integrated with LNC-MEG3 gene.
2. Lentiviral as claimed in claim 1, described carrier is pCDH-EF1-MCS-T2A-copGFP-LNC-MEG3.
3., containing the host cell of the Lentiviral described in claim 1-2, wherein said MEG3 gene is process LAN.
4. host cell as claimed in claim 3, described cell be selected from 293T cell, CML-CD34+ cell KCL22 cell, K562 cell.
5. the application of the expression vector described in claim 1-2 in Tumor suppression generating process.
6. prepare a method for Lentiviral, it is characterized in that with slow virus carrier system pCDH-EF1-MCS-T2A-copGFP, and build based on packaging plasmid pcDNA3.0-LNC-MEG3, described method comprises the steps:
(1) pcr amplification of MEG3 is carried out with following primer:
MEG3V1-FXbaI:TGCTCTAGA GCAGAGAGGGAGCGCGCCTTG
MEG3V1-RNotI:ATTTGCGGCCGC CACATTTATTGAGAGCACAGTG
(2) double digestion is carried out to PCR primer and lentiviral vectors carrier pCDH-EF1-MCS-T2A-copGFP respectively with restriction enzyme Xba I, Not I;
(3) with T4DNA ligase enzyme, goal gene is connected with carrier;
(4) transformed competence colibacillus cell also identifies recombinant vectors by bacterium colony PCR, and the primer is:
Forward primer: people MEG3-f:5 '-GCTCTACTCCGTGGAAGCAC-3 '
Reverse primer: people MEG3-R:5 '-CAAACCAGGAAGGAGACGAG-3 '.
7. by the Lentiviral prepared by claim 6.
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