CN102703503A - Human and mammal cell attachment expression vector and application thereof - Google Patents
Human and mammal cell attachment expression vector and application thereof Download PDFInfo
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Abstract
The invention provides a human and mammal cell attachment expression vector. The expression vector contains a nuclear matrix attachment region sequence, wherein the nuclear matrix attachment region sequence is shown as Seq ID No. 1 or is a nucleotide sequence which is homologous with the sequence by over 95 percent. The vector can mediate stable, high-efficiency and lasting expression of exogenous genes in mammal cells, has high safety performance, can be used for gene therapy and has broad market prospect.
Description
Technical field
The present invention relates to the genetically engineered field, specifically, relate to a kind of mankind and mammalian cell and adhere to expression vector and application thereof.
Background technology
Gene therapy technology is meant and can normal gene or medicative dna sequence dna be imported defective or the performance therapeutic action of target cell to correct gene through certain way, thereby reaches the purpose of treatment disease.Since early 1990s is successfully carried out the clinical trial of gene therapy first, accomplished thousands of routine gene therapy clinical trials so far, wherein expression vector plays a part crucial in gene therapy.The mode that gets into host cell according to carrier is divided into virus vector and plasmid vector.Be widely used in clinical owing to the transfection efficiency of virus vector is higher than plasmid vector; Yet rely on the interaction of virus vector and host cell proteins film to get in the host cell body during virus vector (like adenovirus, adeno-associated virus, retroviral vector) transfection host cell, exist potential carinogenicity, autoimmunization originality and/or cause cell pathology change etc.Whether have the ability of duplicating according to its carrier itself, can virus vector be divided into two types: integrative vector and nonconformity carrier.Integrative vector can cause the generation of inserting sudden change or causing disease owing to insert the position effect of gene.Therefore, foreign gene can be stablized in order to make, safe, the expression that continues, demands developing a kind of novel non-viral attachment carrier urgently.
(matrix attachment region MAR) is meant that after restriction enzyme digestion still attached to the dna sequence dna on the nuclear matrix, length is 200～2000bp to matrix attachment region.Discover that the MAR sequence does not have homologous sequence but has typical constructional feature, as be rich in AT base pair (＞70%), contain " consensus sequence " and the topology isomerase II binding site of several weak points.It plays an important role in the regulating transgene expression of mammalian cell: cis-acting elements is provided, has the function of replication origin and promotor; Avoid apparent silence, improve the transgene expression level in the carrier.
Summary of the invention
The purpose of this invention is to provide a kind of mankind and mammalian cell and adhere to expression vector and application thereof.
In order to realize the object of the invention; A kind of mankind of the present invention and mammalian cell adhere to expression vector; In said expression vector, contain the matrix attachment region sequence, said matrix attachment region sequence shown in Seq ID No.1, or with this sequence 95% above homologous nucleotide sequence.
Aforementioned expression vector, its carrier that sets out is the mankind and mammalian cell expression vector.Be preferably pEGFP-N1, pEGFP-N3, pEGFP-C1, pEGFP-C2, pEGFP-1, pEYFP-N1, pEYFP-C1, pDsRedN1, pDsRedC1, pDsRed2N1, pCDNA3.1 (+), pCDNA3.1 (-), pCDNA3.1 (neo+) or pCDNA3.1 (zeo+) etc.Said matrix attachment region sequence is inserted into the MCS place of said expression vector.
The present invention also provides and contains the host cell that the said mankind and mammalian cell adhere to expression vector.
The present invention also provides the said mankind and mammalian cell to adhere to the application of expression vector in mediate foreign gene is expressed; It is that exogenous gene cloning to the described mankind and mammalian cell adhered in the expression vector; And the carrier that builds changed in the host cell, duplicate expression with the host.Wherein, said foreign gene is positioned at the upper reaches of matrix attachment region.Said host cell is the mankind and mammalian cell, as Chinese hamster ovary (CHO) cell, mouse brain neuroblastoma cell (Neuro-2a, N-2a) etc.
The non-viral attachment carrier of the mankind provided by the invention and mammalian cell can mediate foreign gene stable, efficient, persistent expression in mammalian cell, and safety performance is good.Compare with other carrier, have the following advantages:
(1) matrix attachment region of the present invention (MAR) sequence than in other attachment carrier the MAR sequence length that uses shorter, make carrier have the ability of holding foreign gene more greatly.
(2) the attachment carrier transgenic of MAR sequence of the present invention mediation can be in Chinese hamster ovary celI efficient long-term expression.
(3) the attachment carrier of MAR sequence mediation of the present invention can efficiently express in the tumour cell midium or long term, can connect goal gene and be used for the targeted therapy tumour.
Description of drawings
Fig. 1 is a pEGFP-C1 carrier structure synoptic diagram.
Fig. 2 is a pEGFP-C1-MAR vector construction schema.
Fig. 3 is carrier pEGFP-C1-MAR restriction enzyme digestion and electrophoresis figure; Wherein, 1: digested plasmid not; The 2:BamHI single endonuclease digestion; The 3:KpnI/BamHI double digestion; M:DL5000.
Fig. 4 forms the positive monoclonal cell strain for the transfection group cell; Wherein, the Chinese hamster ovary celI after the a:G418 screening transfection in 30 days forms the monoclonal cell strain; N-2a cell after the b:G418 screening transfection in 30 days forms the monoclonal cell strain.
Fig. 5 is transfection plasmid and reduction plasmid and the preceding plasmid enzyme restriction interpretation of result of transfection; Wherein, M:DL5000; 1: transfection plasmid KpnI/BamHI double digestion; 2: transfection plasmid BamHI single endonuclease digestion; Reduction plasmid KpnI/BamHI double digestion in the 3:CHO cell; Reduction plasmid BamHI single endonuclease digestion in the 4:CHO cell.
Fig. 6 is the expression situation of transfection pEGFP-C1-MAR reporter gene egfp in the Chinese hamster ovary celI; Wherein, a:G418 screening stablizing CHO monoclonal cell 30 days (visible light); The expression (fluorescence) that 30 days egfp of CHO clone cell are stablized in the b:G418 screening; C:G418 screening stablizing CHO monoclonal cell 120 days (visible light); The expression (fluorescence) that 120 days egfp of CHO clone cell are stablized in the d:G418 screening; Scale: 20 μ m.
Fig. 7 is the fluorescence in situ hybridization experimental result, and the pEGFP-C1-MAR carrier adheres to low copy number in Chinese hamster ovary celI.
Fig. 8 is the structural representation of recombinant vectors pMP16.
Fig. 9 detects the expression in the N-2a cell of P16 gene after transfection for Western blot; Wherein, 1: unconverted N-2a groups of cells; 2: transform the N-2a cell.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
1.1 material and method
1.1.1pEGFP-C1 carrier (Fig. 1) is the expression vector of the mankind and mammalian cell, contains reporter gene EGFP on it, available from U.S. Clontech company.
Chinese hamster ovary (CHO) cell is available from Chinese Academy of Sciences's Shanghai cell bank.
1.1.2MAR sequence is synthetic
The characteristic element that contains 2150bp MAR sequence (GenBank:L22754.1) (for example the being rich in the AT sequence) shearing of human MAR sequence 2200bp (GenBank:M83137.1) and human beta-globin is spliced into 387bp, the short MAR sequence of synthetic shown in Seq ID No.1.
1.1.3 endonuclease reaction
Utilize KpnI and BamHI double digestion MAR characteristic sequence and pEGFP-C1 carrier, it is following that enzyme is cut system: plasmid 25 μ l (1 μ g), and 20 * K buffer, 5 μ l, each 2.5 μ l (20U/ μ l) of enzyme mend ultrapure water to 100 μ l, behind the mixing, 37 ℃ of water-bath 4h.After enzyme cut, 1.5% agarose gel electrophoresis detected enzyme and cuts situation, and gel reclaims MAR characteristic sequence and linear pEGFP-C1 plasmid.
1.1.4 connect
The linear pEGFP-C1 plasmid that reclaims is connected reaction system 10 μ l, T according to 1: 5 (mol ratio) with dna sequence dna
4Ligase enzyme 1 μ l, 16 ℃ of reaction overnight.
1.1.5 transform
In competent cell E.coli DH5 α, add 10 μ l and connect product and transform, be seeded in the substratum that contains kantlex 37 ℃ and spend the night picking list bacterium colony.37 ℃ are shaken bacterium 300r/min and spend the night employing SDS alkaline lysis method of extracting recombinant plasmid pEGFP-C1-MAR; Get 10 μ l recombinant DNAs and identify with the KpnI single endonuclease digestion with KpnI/BamHI double digestion and 6 μ l DNA, send the order-checking of Beijing Ying Jun company, the result shows that the expression vector of structure is correct.
PEGFP-C1-MAR vector construction schema is as shown in Figure 2, and the restriction enzyme digestion and electrophoresis figure of recombinant plasmid pEGFP-C1-MAR is as shown in Figure 3.
1.1 utilize Suo Hua-sofast cationic polymers infection protocol will not have intracellular toxin pEGFP-C1-MAR plasmid transfection Chinese hamster ovary celI, Chinese hamster ovary celI is to cultivate in the 800 μ g/mlG418DMEM perfect mediums at final concentration.
Form positive colony 1.2 treat the positive group of Chinese hamster ovary celI transfection, after the whole death of control wells cell, change in the DMEM perfect medium that contains 400 μ g/ml concentration G418 and cultivate.
1.3 when Chinese hamster ovary celI reached 70～90%, collecting cell utilized the blood counting chamber counting process to count, and was roughly 1 cell with the cell count of serum-free DMEM substratum dilution in cell count makes every hole, and then added an amount of perfect medium.
1.4 observe the growing state of monoclonal cell, still keep selective pressure with the DMEM substratum of half G418 concentration, when treating that monoclonal cell density reaches hole internal volume 1/3, be transferred to after the trysinization and carry out enlarged culturing in the culturing bottle.
The plastidogenetic positive monoclonal cell strain of transfection group is shown in Fig. 4 a.
1.1 utilize the Hirt cracking process to extract the attachment plasmid in two kinds of stable monoclonal cells: in the cell of collecting, add Hirt lysis buffer 2ml, room temperature leaves standstill 20min; The 5mol/L NaCl that adds 1/4 volume (0.5ml), 4 ℃ are spent the night.Next day, the centrifugal 40min of 15000rpm.Get supernatant, add RNase A (10mg/mL) 125 μ l in 37 ℃ of water-bath 60min; With phenol chloroform extracting 2 times.Get supernatant, the Virahol that adds 5ml-20 ℃ of precooling places-20 ℃ to spend the night.Next day, the centrifugal 30min of 12000rpm.Absolute ethanol washing 2 times behind the air drying, adds 10 μ l elution buffers dissolving attachment plasmid.
1.2 getting the competent cell 250 μ l of prepared fresh under the sterile state adds 10 μ l attachment plasmids and transforms.Add the SOC solution 800 μ l of 37 ℃ of preheatings during recovery, place 37 ℃ of shaking table 250rpm shaking culture 2h.Get the bacterium liquid 300 μ l of above-mentioned conversion, coat the whole 50 μ g/ml Kan that contain equably
+And 37 ℃ of preheating agar plate surface, grow until positive transformant.
1.3 the picking monoclonal cell shakes bacterium and extracts plasmid, the double digestion system is following: plasmid 10 μ l, and 20 * K buffer, 1 μ l, each 0.5 μ l of KpnI/BamHI mends ultrapure water to 20 μ l.Fully behind the mixing, behind 37 ℃ of water-bath 6h in 1.5% sepharose electrophoresis detection.
The transfection plasmid is as shown in Figure 5 with reduction plasmid and the preceding plasmid enzyme restriction interpretation of result of transfection.
1.1 fluoroscopic image analysis
After G418 resistance screening CHO (Fig. 6) stablizes monoclonal cell 30d and 120d, the expression of visual report gene egfp and gather image analysis under the fluorescent microscope.
1.2 the preparation of culturing cell Metaphase Chromosome
When the fusion of clone reaches about 75～90% the time, in the substratum cell of every 5ml, add 40 μ l NST-757s, continue to cultivate 2h and collecting cell.Hypotonic: add the 0.075mol/L KCl solution 10ml of 37 ℃ of water-bath preheatings, 37 ℃ of hypotonic 30min of water-bath make the abundant dispersion suspension of cell in solution.Pre-fix: in centrifuge tube, add the 1.5ml stationary liquid and blow evenly with suction pipe gently, pre-fix 10min.The centrifugal 5min of 2000rpm abandons supernatant.Fixing I: slowly add stationary liquid 6ml along tube wall, blow and beat into cell suspension with suction pipe after, 37 ℃ of water-bath 20min.The centrifugal 10min of 1000rpm abandons supernatant.Fixing II: operation steps is with fixing I, stays supernatant 0.5～1ml after centrifugal, blows and beats into cell suspension with suction pipe.Drip sheet: draw the small amounts of cells suspension with suction pipe, height 30～40cm vertically drops on the slide glass of precooling, and room temperature is dried.Aging: the karyomit(e) slide places 37 ℃ to wear out 1～7 day.Slide places 60 ℃ of incubation 3～4h before using.
1.3 fluorescence in situ hybridization experiment
3～5ml sex change liquid is placed 74 ± 1 ℃ of water-baths or hybridization instrument balance 30min at least, slide is immersed 2～5min in the hybridization solution.Gradient dehydration 1min.Probe is placed sex change in the PCR appearance, and program is made as: 77 ℃ of 5min, and 37 ℃ of 2min, on slide, hybridization spends the night (12～18h) with the probe Rubber glue mounting after the sex change.Slide is placed 72 ℃ of 30min at least, throw off the glue on the slide gently.72 ℃, washing 2min; Normal temperature washing 2min.Each 1min of gradient dehydration, dehydration back air drying.Redye and observations: add diamino-phenyl indoles (DAPI) 20 μ l and redye, add deckglass.Place dark place 20min, the fluorescence microscope result also gathers image (Fig. 7).The result shows, the pEGFP-C1-MAR carrier adheres to low copy number in Chinese hamster ovary celI and exists, and carrier molecule is attached on the Metaphase Chromosome, and the copy number of carrier molecule is approximately 4 copy/cells, does not have specific binding site.
Embodiment 5 contains adhering to expression vector and pressing down the knurl functional analysis of people P16 cancer suppressor gene
1.1 the clone of people P16 cancer suppressor gene
P16 suicide gene sequence (Genbank:AB060808) design PCR primer P1 and P2 according to the people; Wherein primer P1 comprises NheI and restriction enzyme site; Primer P2 comprises the HindIII restriction enzyme site, and primer sequence is (italic is a gene order, and underscore is a restriction enzyme site) as follows:
P15′-GC
GCTAGCATGGAGCCTTCGGCTGACTTT-3′，
P25′-CG
AAGCTTTCAATCGGGGATGTCTGAGGGA-3′；
Get human peripheral 100 μ l,, extract total RNA by the Trazol test kit operation instructions of extracting RNA, get a certain amount of total RNA, after its integrity of 1.0% denaturing formaldehyde agarose gel electrophoresis evaluation ,-70 ℃ of preservations, subsequent use; In 50 μ L reaction systems; Add dNTP1 μ L (10mmol/L) respectively; RNA l μ g, concentration is P1, P2 primer each 1 μ L and AMV-Taq archaeal dna polymerase 1 μ L (5U/ μ L) of 20 μ mol/L, AMV ThermoScript II 1 μ L (5U/ μ L); Carry out the RT-PCR reaction according to One Step PCR Kit (AMV) test kit operation instructions, become the reaction conditions of cDNA following the mRNA reverse transcription: 50 ℃ of 30min; Carry out the PCR reaction by following condition then: 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 60s, totally 30 circulations.Get PCR product 10 μ L and carry out the detection of 1.5% agarose gel electrophoresis, it is 471bp that the result amplifies big or small fragment, conforms to the expection size; Reclaim target DNA with the dna gel purification kit, the order-checking back is the result show, the P16 gene cDNA sequence of dna fragmentation that amplifies and GenBank login is in full accord.
1.2 adhere to the structure of expression vector pMP16
With NheI and HindIII double digestion pcr amplification product, the pEGFP-C1-MAR plasmid that makes up with NheI and HindIII double digestion embodiment 1 simultaneously adopts conventional enzyme blanking method to carry out enzyme and cuts, gel excision EGFP gene; P16 goal gene after subsequently the enzyme switchback being received is connected by 1: 5 (mol ratio) with shape material grain pEGFP-C1-MAR, and 16 ℃ of connections are spent the night; To connect then in the product adding E.coli DH5 α competent cell suspension and transform; Cell suspension inoculation after transforming is being contained 37 ℃ of overnight cultures on the LB flat board of kantlex; Picking list bacterium colony succeeding transfer culture, and carry out double digestion (NheI and the HindIII) checking of recombinant plasmid vector; Choose enzyme and cut the correct plasmid of checking, carry out sequence verification, the right-on carrier called after of target gene sequences pMP16 (Fig. 8).By conventional plasmid process for extracting extracting and purifying pMP16 plasmid, measure OD
260Plasmid concentration.
1.3 the foundation of stable transfected cells strain
Mouse brain neuroblastoma cell N-2a is available from Chinese Academy of Sciences's Shanghai cell bank.Utilize Suo Hua-sofast cationic polymers infection protocol will not have intracellular toxin pMP16 plasmid transfection N-2a cell, in the G418 DMEM perfect medium of final concentration 600 μ g/ml, cultivate.Treat that the positive group of N-2a cell transfecting forms positive colony, after the whole death of control wells cell, changes in the DMEM perfect medium that contains 300 μ g/ml G418 and cultivates.When N-2a cytogamy degree reaches 70～90% respectively; Collecting cell; Utilize the blood counting chamber counting process to count, be roughly 1 cell with the cell count of serum-free DMEM substratum dilution in cell count makes every hole, and then add an amount of perfect medium.Observe the growing state of monoclonal cell, still keep selective pressure, when treating that monoclonal cell density reaches hole internal volume 1/3, be transferred to after the trysinization and carry out enlarged culturing in the culturing bottle with the DMEM substratum of half G418 concentration.The plastidogenetic positive monoclonal cell strain of transfection group is shown in Fig. 4 b.
1.4Western the blot method detects the proteic expression of transfectional cell P16
The collecting cell supernatant is with 15KD aperture dialysis membrane dialysis 2h, 1/10 of simmer down to original volume; In the sample of collecting, add isopyknic SDS-PAGE albumen sample-loading buffer, boiling water bath heating 5min is with abundant metaprotein; The centrifugal 2min of 12000rpm gets supernatant and carries out the SDS-PAGE electrophoresis.At first prepare 15% separation gel and 5% spacer gel, install glue-pouring device, record 15% separation gel, the back suction is solidified in water seal under the room temperature, fill spacer gel again, inserts comb, treats to take out comb after the spacer gel room temperature is solidified, and assembles electrophoresis apparatus.Each histone sample is mixed the back go up appearance with tetrabromophenol sulfonphthalein; Use low voltage 80V constant voltage electrophoresis during glue on the upper strata during electrophoresis, treat that tetrabromophenol sulfonphthalein enters into separation gel after, use high-voltage 120V constant voltage electrophoresis; When continuing electrophoresis to tetrabromophenol sulfonphthalein arrival separation gel bottom; Stop electrophoresis, peel separation glue, and corner cut marking.Adhesive tape is cut to suitable size, and the same big filter paper with gel of cutting and nitrocellulose filter immerse 10min in the transfering buffering liquid together simultaneously, and membrane-transferring device is pressed negative pole, filter paper, gel, nitrocellulose filter, filter paper, anodal stack from bottom to up; And accurately alignment, two filter paper contact about avoiding, and carefully remove bubble; To avoid short circuit; Place the electrophoretic blotting groove, energized, 200mA electrotransfer 90min.After the end, deenergization takes out nitrocellulose filter, spends the night with 4 ℃ of sealings of 5% skim-milk, with the blocking-up non-specific binding.Take out nitrocellulose filter, with TBS washing 3 times, each 10min places one anti-(the mouse P16 of rabbit Chinese People's Anti-Japanese Military and Political College polyclonal antibody) of dilution in 1: 200 respectively with film, slowly shakes 2h under the room temperature; Abandon one and resist, film with TBS washing 3 * 10min, is placed two anti-(diluting at 1: 1000) with the TBST dilution HRP mark that contains 5% (w/v) degrease milk powder, incubated at room 1h with film; Abandon two anti-ly, film with TBS washing 3 * 10min, is dripped the ECL luminescence reagent after blotting excessive moisture on film, room temperature effect 5min puts into magazine and is exposed on the X line film, after development and photographic fixing, preserves.Use β-actin antibody as last appearance contrast.
Western blot result (Fig. 9) shows, the specific band that transfection P16 group appears; Above-mentioned band does not all appear in untransfected group and cell control group in the corresponding position, show that pMP16 can mediate exogenous P16 gene successful expression target protein in the N-2a cell.
1.5 flow cytometer detects apoptosis
To logarithmic phase, collecting cell, PBS clean 2 times with transgenic N-2a cell cultures, with 1 * binding buffer liquid adjusting cell to 1 * 10
6Individual/ml, get cell 100 μ l in the streaming pipe, add 10 μ l Annexin-V-PE mixing on ice, lucifuge 15min.Add 1 * binding buffer liquid, 380 μ l again, add 10 μ l 7-AAD again, the detected by flow cytometry apoptosis rate.The experiment triplicate.The streaming result that the FCM of PI staining detects is as shown in table 1: transgenic pMP16 group N-2a apoptosis rate is 19.28 ± 1.56%, apparently higher than control group apoptosis rate 2.78 ± 0.20 (P＜0.01).
Table 1P16 suicide gene system is to the result of N-2a cell apoptosis
Annotate: a.P＜0.01, b.P＜0.05
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (7)
1. the mankind and mammalian cell adhere to expression vector, it is characterized in that, contain the matrix attachment region sequence in the said expression vector, said matrix attachment region sequence shown in Seq IDNo.1, or with this sequence 95% above homologous nucleotide sequence.
2. expression vector according to claim 1 is characterized in that, its carrier that sets out is the mankind and mammalian cell expression vector.
3. expression vector according to claim 1 and 2; It is characterized in that its carrier that sets out is pEGFP-N1, pEGFP-N3, pEGFP-C1, pEGFP-C2, pEGFP-1, pEYFP-N1, pEYFP-C1, pDsRedN1, pDsRedC1, pDsRed2N1, pCDNA3.1 (+), pCDNA3.1 (-), pCDNA3.1 (neo+) or pCDNA3.1 (zeo+).
4. contain the host cell that each described mankind of claim 1-3 and mammalian cell adhere to expression vector.
5. each described mankind of claim 1-3 and mammalian cell adhere to the application of expression vector in mediate foreign gene is expressed; It is characterized in that; Exogenous gene cloning is adhered in the expression vector to each described mankind of claim 1-3 and mammalian cell; And the carrier that builds changed in the host cell, duplicate expression with the host.
6. application according to claim 5 is characterized in that said foreign gene is positioned at the upper reaches of matrix attachment region.
7. application according to claim 5 is characterized in that, said host cell is the mankind and mammalian cell.
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| CN105039411A (en) * | 2015-08-27 | 2015-11-11 | 新乡医学院 | Attached type lentiviral vector as well as preparation method and application thereof |
| CN106544362A (en) * | 2016-12-06 | 2017-03-29 | 新乡医学院 | A kind of mammalian cell based on cHS4 elements adheres to body expression vector, expression system, preparation method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103642838A (en) * | 2013-11-29 | 2014-03-19 | 新乡医学院 | Human and mammal cell expression vector and application thereof |
| CN105039411A (en) * | 2015-08-27 | 2015-11-11 | 新乡医学院 | Attached type lentiviral vector as well as preparation method and application thereof |
| CN105039411B (en) * | 2015-08-27 | 2019-01-18 | 新乡医学院 | Attached type slow virus carrier, preparation method and application |
| CN106544362A (en) * | 2016-12-06 | 2017-03-29 | 新乡医学院 | A kind of mammalian cell based on cHS4 elements adheres to body expression vector, expression system, preparation method and application |
| CN106544362B (en) * | 2016-12-06 | 2019-04-09 | 新乡医学院 | A kind of mammalian cell attachment body expression vector, expression system, preparation method and application based on cHS4 element |
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