CN105039404A - Dual regulation control recombinant adeno-associated virus package system for targeted tumor - Google Patents
Dual regulation control recombinant adeno-associated virus package system for targeted tumor Download PDFInfo
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Abstract
The invention discloses a dual regulation control recombinant adeno-associated virus package system for a targeted tumor. Auxiliary adenovirus E1a, E1b, E2a, E4 and VARNA genes necessary for adeno-associated virus propagation in the package system are effectively controlled by a telomere reverse transcriptase (hTERT) promoter, a Rep gene of a packaging plasmid is effectively controlled by a hexokinase (HKII) promoter, the adeno-associated virus package system is only expressed in a target cell with hTERT activity and HKII activity, and the modified adeno-associated virus package system has very high tumour cell targeting. The adeno-associated virus package system provides foundation for oncolytic gland-related virus, and has great promotion action for tumor biotherapy.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of two regulation and control recombinant adeno-associated virus packaging systems of target tumor.
Background technology
Malignant tumour is that current serious affects one of human health, the major disease threatening human life, and current clinical treatment mainly adopts radiotherapy and chemotherapy.But some malignant tumour is insensitive to radiotherapy, easily produces resistance to chemotherapy, and this treatment plan is usually with obvious toxic action.Therefore, research selectively killing tumour cell and not affect Normocellular method extremely important to oncotherapy, the method of this selectively killing tumour cell depends on the specific marker of tumour cell, and its curative effect also depends on whether this specific marker is strictly limited in tumour cell.
Telomere is the structure of eukaryotic cell protection end of chromosome, and normal cell often divides once, and telomere will shorten, and can cause necrocytosis when telomere foreshortens to a certain degree.And when cell cancerates, its Telomerase is activated, the shortening of telomere caused by tumour cell division is extended further, thus remain chromosomal stable, cell evasion death is immortalized, finally cause tumour cell constantly to rise in value, therefore Activation of Telomerase occurs and its keying action in evolution at tumour cell.Telomerase is all positive at about 90% tumour cell, and thumping majority normal cell Telomerase is feminine gender, and therefore Telomerase can be used as a kind of characteristic marker of tumour cell.Up to the present, the Telomerase of people is made up of three parts: 1) RNA component (hTERC); 2) telomere enzyme binding protein (TEP1); 3) telomere reversed transcriptive enzyme (hTERT).Nearest research shows that telomere reversed transcriptive enzyme (hTERT) plays a decisive role in telomerase activation, and in thumping majority tumour cell or tumor cell line, telomere reversed transcriptive enzyme is high level expression, and does not express or low expression level in normal cell.
Normocellular energy is mainly derived from glucose, in the well-off situation of oxygen, normal cell tissue is mainly through glycolysis-and mitochondrial respiratory two steps, glucose oxidase is become carbonic acid gas and water, for cell biological activity provides energy, time oxygen is under-supply, rely on glycolysis-supplying energy.Even if malignant cell is under the well-off condition of oxygen, still high glycolysis-ratio is had to be a specific performance of malignant tumour energy metabolism aspect, before 80 years, first OttoWarburg finds this phenomenon in liver cancer cell, and this phenomenon is called as Warburg effect.Hexokinase is the attemperator of glycolysis-and energy metabolism, and in malignant cell, hexokinase II (HKII) expresses and increases.HKII is the tumour cell glycolytic ferment of current most study.The people such as Ko are shown by experimentation on animals, HKII Pharmacological inhibitors (3-BrPA) local and systemic administration can kill most of liver transplantation knurl and pulmonary metastases, but to all normal liver tissues of cancer and the harmless effect of other main organs, HKII is suppressed to have obvious lethal effect (KoYH equally to the tumour cell of chemotherapy resistance, PedersenPL, GeschwindJF.GlucosecatabolismintherabbitVX2tumormodelfor livercancer:characterizationandtargetinghexokinase [J] .CancerLett.2001, 173 (1): 83-91).The height of HKII in malignant tumour is expressed, and is the biological property of malignant tumour, is also the needs of tumour existence.
In recent years, due to the develop rapidly of virusology, molecular biology and oncology, people have completed the examining order of multiple viral full gene group, and comparatively detailed research has been carried out to the structure of its gene and function thereof, can effectively transform virogene, improved virus effectively strengthens the infection of tumour cell, the ability that copies propagation and dissolved cell, and even disappears to this reduced capability of normal cell, and therefore this virus can carry out copy choice in tumour cell.The most deep oncolytic virus of current research comprises I herpes simplex virus type-1 (HSV-1) and adenovirus (ADV) etc.Adeno-associated virus (AAV) be a class small, without tunicle and the tiny single-stranded DNA viruses with icosahedral structure of virus, the features such as AAV has that security is good, host range is wide, immunogenicity is low, the expression alien gene time is long in vivo, be regarded as one of the most promising gene rotaring carrier, but its application in oncolytic is ground to make internal disorder or usurp and be yet there are no report.
Build restructuring AAV and need AAVITR sequence (reversing terminal repeat), Rep and Cap gene, host cell and helper virus.AAV carrier used in gene therapy research is at present most to be transformed by 2 type AAV, contains the signal copying, pack, save and integrate in the ITR sequence of AAV-2; Exogenous gene expression box can replace the encoding sequence of AAV completely, Rep and Cap gene product then can provide by another plasmid is trans; It is adenovirus that AAV copies conventional helper virus, and the subsidiary function of adenovirus is provided by E1a, E1b, E2a, E4 and VARNA gene.
Summary of the invention
The present invention utilizes Telomerase and glycolysis-as the specific marker of tumour cell, adopt three plasmid co-transfection method transfectional cell encapsidated adenovirus correlated virus, wherein Adv-hTERT-Ep-E1a helper plasmid carries adeno-associated virus and breeds necessary helper adenovirus E1a, E1b, E2a, E4 and VARNA gene, pHKII-AAV-RC packaging plasmid carries adeno-associated virus and breeds necessary Rep and Cap gene, and expression vector plasmid carries adeno-associated virus and copies necessary ITR sequence.Simultaneously, application telomere reversed transcriptive enzyme (hTERT) promotor regulates and controls E1a, E1b, E2a, E4 and VARNA gene, hexokinase II (HKII) promotor regulates and controls Rep gene, thus this adeno-associated virus packaging system can only be had in the target cell of active and hexokinase II (HKII) activity of telomere reversed transcriptive enzyme (hTERT) at the same time express, the adeno-associated virus packaging system after this modification has high tumour cell targeting.
Two regulation and control recombinant adeno-associated virus packaging systems of a kind of target tumor disclosed by the invention, it is characterized in that: in this packaging system, adeno-associated virus breeds the effective control of necessary helper adenovirus E1a, E1b, E2a, E4 and VARNA gene by telomere reversed transcriptive enzyme (hTERT) promotor, the Rep gene of packaging plasmid is subject to effective control of hexokinase II (HKII) promotor.
Two regulation and control recombinant adeno-associated virus packaging systems of above-mentioned a kind of target tumor, is characterized in that this packaging system is made up of following plasmid:
(1) the adeno-associated virus helper plasmid Adv-hTERT-Ep-E1a of hTERT promoter regulation;
(2) the adeno-associated virus packaging plasmid pHKII-AAV-RC of HKII promoter regulation
(3) glandular associated virus expression vector plasmid.
Two regulation and control recombinant adeno-associated virus packaging systems of above-mentioned a kind of target tumor, is characterized in that employing three plasmid co-transfection method transfectional cell encapsidated adenovirus correlated virus.
Two regulation and control recombinant adeno-associated virus packaging systems of above-mentioned a kind of target tumor, is characterized in that described cotransfection method is electroporation, calcium phosphate precipitation, lipofection, multiple cationic substance or virus-mediated transfection method.
Two regulation and control recombinant adeno-associated virus packaging systems of above-mentioned a kind of target tumor, is characterized in that described cotransfection method is lipofection.
Two regulation and control recombinant adeno-associated virus packaging systems of above-mentioned a kind of target tumor, is characterized in that described packing cell is any one in HEK293 cell, human liver cancer cell (HepG2), human lung carcinoma cell (A549), MCF-7 (human breast cancer cell) and bronchial airways epithelial cell (NHBEC).
Beneficial effect of the present invention is: the open adeno-associated virus packaging system of the present invention can only have in the target cell that telomere reversed transcriptive enzyme (hTERT) is active and hexokinase II (HKII) is active at the same time to be expressed, adeno-associated virus packaging system after this modification can in tumour cell normal expression, pack out adeno-associated virus, and express extremely low in normal cell, there is high tumour cell targeting; Adeno-associated virus packaging system disclosed by the invention is laid a good foundation for studying oncolytic adeno-associated virus, has great promoter action to biotherapy tumour.
Accompanying drawing explanation
Fig. 1 is pHKII-AAV-RC plasmid construction procedure chart;
Fig. 2 is virus titer result figure.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (calculating three editions, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
Embodiment 1 builds the adeno-associated virus helper plasmid of hTERT promoter regulation
Build containing telomere pol gene promoter plasmid Adv-hTERT-Ep-E1a (concrete grammar see Zhang Qi, virus of a kind of proliferated specifically inside tumor cell of high expression antioncogene and uses thereof, CN1219055C).
Embodiment 2 builds the adeno-associated virus packaging plasmid of HK-II promoter regulation
Build and (synthesized by the raw work in Shanghai containing HK-II promoter plasmid pUC-HK-II, specifically see MathupalaSP.Glucosecatabolismincancercells.Isolation, sequence, andaetivityofthepromoterfortypeIIhexokinase [J] .BiolChem, 1995,270:16918-16925), with the linearizing of HindIII single endonuclease digestion, and reclaiming pUC-HK-II plasmid enzyme restriction product, detailed step is see (TAKARA glue reclaims test kit).
By pAAV-RC plasmid (Stratagene company) restriction enzyme XmaI and XbaI double digestion (TAKARA), glue reclaims the fragment of clip size 4253bp.Employing fills connection test kit, the enzyme of pAAV-RC plasmid is cut back to close fragment, insert in linearizing pUC-HK-II plasmid through connecting, (detailed step is see TAKARA for sequence verification, BluntingKinationLiagtion (BKL) Kit), its building process as shown in Figure 2, constructs plasmid pUC-HK-II-AAV-RC, called after pHKII-AAV-RC.
Embodiment 3 builds pAAV-IRES-ZsGreen glandular associated virus expression vector
PAAV-IRES-ZsGreen vector construction with plasmid pLVX-IRES-ZsGreen (En Wei bio tech ltd, Shenzhen hundred) for template, PCR method amplification IRES-ZsGreen, HindIII and SpeI is introduced in upstream primer IZF, introduce BgIII in downstream primer IZR, design PCR primer sequence is as follows:
IRES-ZsGreen-F(IZF)：5’-GTGCAAGCTTACTAGTCTAGTGCCCCTCTCCCT-3’(SEQIDNO.1)
IRES-ZsGreen-R(IZR)：5’-CTAGAGATCTTTCAGGGCAAGGCGGAG-3’(SEQIDNO.2)
Sepharose reclaims the object band of 1283bp.Product and vector plasmid pAAV-MCS (Stratagene company) is reclaimed with HindIII and BglII double digestion glue, after two digestion products with cohesive terminus being connected with ligase enzyme, transformed competence colibacillus DH5 α (TAKARA) bacterial strain, Amp plate screening positive colony, identify with HindIII and BglII double digestion, identify that correct recombinant clone carries out sequence verification, verify correct plasmid markers be pAAV-IRES-ZsGreen (Detailed operating procedures is see the packaging of Chen Ruyi .hIL-2 and ZsGreen double gene coexpression adeno-associated virus and qualification [J]. Zhejiang University of Traditional Chinese Medicine's journal, 2014, 38 (7): 875-880).
Embodiment 4 tumour-specific adeno-associated virus packaging system virus packaging
The present invention uses HEK293 cell, human liver cancer cell (HepG2), human lung carcinoma cell (A549), MCF-7 (human breast cancer cell) and bronchial airways epithelial cell strain (NHBEC) (above-mentioned cell is all purchased from Chinese Academy of Sciences's Shanghai cell bank), respectively the tumour-specific adeno-associated virus packaging system of transfection structure.Each cell encapsidated adenovirus correlated virus detailed step method is all with reference to following 293 cell packaging steps.
1, the recovery of 293 cells
1. set temperature is the water-bath of 37-42 DEG C;
2. check cell bank record, from liquid nitrogen container, take out frozen cell according to record, lose rapidly in water-bath and also rock fast, in 1 ~ 2min, make cell solution dissolve completely as far as possible;
3. cell solution is transferred in 15ml centrifuge tube, and add the perfect medium that 1ml is fresh wherein, centrifugal after mixing, 1000rpm/min, 5min;
4. remove supernatant, add the perfect medium that 5ml is fresh, after mixing precipitation, proceed to 6cm culture dish;
5. culture dish is steadily put into 37 DEG C, 5%CO
2cultivate with in the incubator of 95% relative humidity.
2, AAV packs and concentrates
1. plasmid amplification
The AAV carrier built and packaging, helper plasmid need through a large amount of extracting, and concentration is greater than 1ug/ul, and A260/280 can in order to wrap poison between 1.7-1.8.Using Qiagen to take out greatly, test kit carries out plasmid goes intracellular toxin extracting in a large number.
2. 293 cells are passed
By the substratum exhaustion in cultivation 293 cell T75 bottle, add 0.25% pancreatin that 2mL4 DEG C of refrigerator takes out, make at the bottom of its uniform fold bottle, be placed in 37 DEG C of incubator 3-5min, take out, rock and can find that cell departs from bottom, under it is all shaken, add the 10%DMEM of preheating in 3mL37 DEG C of water-bath, blow and beat with 10mL transfer pipet, blow and beat 6-8 time, do not stay dead angle, transfer pipet can be aimed at training mouth by the more difficult piping and druming of bottle mouth position, and substratum is got the cell that can cover close to bottleneck by little power.Afterwards, by all cells sucking-off, be placed in 15mL centrifuge tube, get the cell after 50ul mixing in 1.5mLeppendorf pipe, add 450ul10%DMEM, be 10 times of dilutions, mixing, gets 10ul cell and counts in tally.Go down to posterity and be designated as first day the same day, if second day carries out transfection, paving 900-1000 ten thousand/T75; If transfection in the 3rd day, paving 350-400 ten thousand/T75.Every bottle of T75 adds 10mL10%DMEM substratum.Transfection observation of cell on same day density, 80-90% completely can carry out transfection.
9. fat transfected HEK 293
Adopt lipofection by phTERT-Helper, pHKII-AAV-RC and pAAV-IRES-ZsGreen cotransfection HEK293 cell.Rotaring redyeing system and reagent use Lipofectamine
tM2000 (invitrogen companies), transfection detailed step is with reference to transfection specification sheets.Rotaring redyeing system is as follows:
4. AAV virus receives poison
1) a dry ice ethanol bath and 37 DEG C of water-baths are prepared;
2) cell producing poison is together collected in the centrifuge tube of a 15ml together with substratum.During collecting cell, cell scrapes in substratum by the certain angle that tilted by culture plate;
3) 1000rpm/min, centrifugal 3 minutes, isolated cell and supernatant, deposited in addition by supernatant, and cell 1mlPBS is resuspended;
4) cell suspending liquid is repeatedly shifted in dry ice ethanol bath and 37 DEG C of water-baths, freeze thawing four times.
5. AAV viral concentration
1) 10,000g centrifugal segregation cell debriss, transfer in a new centrifuge tube by centrifuged supernatant;
2) supernatant of twice collection is mixed, with the removal of impurity of 0.45um frit;
3) add the 1MNaCl of 1/2 volume, 10%PEG8000 solution, mixes, and 4 DEG C are spent the night;
4) the centrifugal 2h of 12000rpm, abandons supernatant, and the appropriate PBS solution of viral pellet is dissolved, and uses 0.22um frit degerming until completely dissolved;
5) add Benzonase nuclease digestion and remove residual plasmid DNA (final concentration is 50U/ml), close upper tube cap, put upside down several times fully to mix, hatch 30 minutes at 37 DEG C;
6) filter with 0.45 μm of filtering head, get filtrate, be concentrated AAV virus, preserve-80 DEG C.
3, virus titer measures
The HEK293 cell of paving 8 six orifice plates, the inoculating cell quantity of every orifice plate is 2x10
5individual/hole, after cultivating 12h, adds 5ul respectively and concentrates the AAV virus obtained.After 48h, mark positive rate with flow cytomery cell GFP, virus titer is by (TU/ml)=(2 × 10
5× cell eGFP+ leads)/virus stock solution used volume computing, it the results are shown in Figure 2.
Result show, adeno-associated virus packaging system disclosed by the invention can in tumour cell normal expression, pack out adeno-associated virus, and expression amount is extremely low in normal cell.Therefore, adeno-associated virus packaging system disclosed by the invention has tumour cell targeting, for research oncolytic adeno-associated virus treatment tumour is laid a good foundation, has great promoter action to biotherapy tumour.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (6)
1. two regulation and control recombinant adeno-associated virus packaging systems of a target tumor, it is characterized in that: in this packaging system, adeno-associated virus breeds the effective control of necessary helper adenovirus E1a, E1b, E2a, E4 and VARNA gene by telomere reversed transcriptive enzyme (hTERT) promotor, the Rep gene of packaging plasmid is subject to effective control of hexokinase II (HKII) promotor.
2. two regulation and control recombinant adeno-associated virus packaging systems of a kind of target tumor according to claim 1, is characterized in that this packaging system is made up of following plasmid:
(1) the adeno-associated virus helper plasmid Adv-hTERT-Ep-E1a of hTERT promoter regulation
(2) the adeno-associated virus packaging plasmid pHKII-AAV-RC of HKII promoter regulation
(3) glandular associated virus expression vector plasmid.
3. two regulation and control recombinant adeno-associated virus packaging systems of a kind of target tumor according to claim 1 and 2, is characterized in that employing three plasmid co-transfection method transfectional cell encapsidated adenovirus correlated virus.
4. two regulation and control recombinant adeno-associated virus packaging systems of a kind of target tumor according to claim 3, is characterized in that described cotransfection method is electroporation, calcium phosphate precipitation, lipofection, multiple cationic substance or virus-mediated transfection method.
5. two regulation and control recombinant adeno-associated virus packaging systems of a kind of target tumor according to claim 3, is characterized in that described cotransfection method is lipofection.
6. two regulation and control recombinant adeno-associated virus packaging systems of a kind of target tumor according to claim 3, is characterized in that described cell is any one in HEK293 cell, human liver cancer cell (HepG2), human lung carcinoma cell (A549) and bronchial airways epithelial cell (NHBEC).
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