CN101182539A - Construction method of DC-SIGN promoter luciferase reporting plasmid - Google Patents

Abstract

The invention discloses a construction method of the DC-SIGN promoter luciferase reported plasmid. By constructing the DC-SIGN promoter segment and the recombinant eucaryon of the luciferase reported vector p GL3-Basic, plasmid DC-Promoter-Pgl3-Basic is expressed and the plasmid expression and activity identification in Hela, Hacat, Wish and HuVEC cell strains; the invention successfully constructs the DC-promoter-pGL3-Basic DC-SIGN promoter luciferase reported plasmid. With the expression and luciferase activity identification in Hela, Hacat, Wish and HuVEC cell strains, the DC-SIGN promoter is proven to express different activities in different cell strains; the activities in the cell strains of HaCat and Hela are comparatively high.

Description

The construction process of DC-SIGN promoter luciferase reporting plasmid

Technical field

The invention belongs to technical field of molecular biology, relate to the construction process of DC-SIGN promoter luciferase reporting plasmid.

Background technology

DC-SIGN is a DCs surface important function molecule, plays an important role in the immunity of organism process.By the antigen presentation effect of DCs, DC is presented to virus, bacterium and the protozoon of organism infection around the effector cell, by directly and indirectly killing and wounding of effector cell, and the immunization of performance body, thus eliminate or suppress to infect the pathogenic micro-organism of body.In this process, the DC-SIGN that is positioned at the DC surface plays central role.The position is known up till now, and DC-SIGN is the acceptor of multiple pathogenic micro-organism, comprises HIV, HCV, CMV, SARS, dengue fever virus, Ebola virus, tubercule bacillus, intestinal bacteria core composition, leishmania or the like.

With HIV is example, DC-SIGN can with the combination of HIV-gp120 albumen high-affinity, this avidity is higher than the intermolecular avidity of HIV-gp120-CD4.DC-SIGN is by identification, combination and carry the HIV particle, and the HIV virion is transported to the secondary draining lymph node at position, place from the periphery infection site, infects the T cell in lymphatic node, thereby further virus is propagated to whole body.In this process, the DC-HIV-CD4+ cell forms a combination, this combination, and the distance of spatially furthered HIV and CD4+T cell, it is involutory that the acceptor of the part of HIV and CD4+T cell is easy to, and increased the chance of CD4+T cell infection.In addition, HIV can also and TLR (the Toll Like Receptor) combination on DCs surface, by the reaction in TLR downstream, make the DCs secrete cytokines, raise the expression of DC-IGN.This explanation DC-SIGN not only is subjected to the regulating effect of internal factor in the expression of DC cell, also is subjected to the influence of extraneous factor.

In external expression model, DC-SIGN mainly is subjected to IL-4 effect regulation and control.From the isolating mononuclearcell of human peripheral, through the combined stimulation of IL-4 and GM-CSF, the perhaps cell strain THP-1 in blood system source, Jurknt etc. are through the stimulation of IL-4 and PMA, can make the active DC-SIGN of these cell expressings, express model as DC-SIGN external.And in vivo, DC-SIGN only shows on the DCs of SM some kind of epiderm skin, and locates at enteron aisle lymphoglandula and placenta etc., expresses limitation relatively.

At eukaryotic cell, gene expression regulation mechanism relative complex, still, the promotor of gene still plays a major role in the expression of gene regulation and control.The DC-SIGN gene promoter area has significance for virus infection, therefore, we at first separate the DC-SIGN gene promoter, and be structured in the luciferase reporting carrier, transfection Hela, Hacat, Wish and HuVEC cell strain by the determination of activity of luciferase, are investigated the activity of DC-SIGN promotor respectively, the biology characteristics of research DC-SIGN are inquired into DC-SIGN expression mechanism and the meaning of DC-SIGN promotor in DC-SIGN expression process.

Summary of the invention

The objective of the invention is: the construction process that DC-SIGN promoter luciferase eucaryon reporter plasmid is provided.

The construction process of DC-SIGN promoter luciferase reporting plasmid of the present invention may further comprise the steps:

(1) with reference to the DC-SIGN promoter sequence of GeneBank AF209479, the a pair of primer of design DC-SIGN promotor, introduce Mlu I and Bgl II recognition site respectively at 5 ' end and 3 ' end: ACGCGT and AGATCT, with PCR method high-fidelity amplification DC-SIGN promoter fragment, and purified pcr product, standby:

(2) DC-SIGN promoter fragment and its expression vector pGL3-basic are carried out double digestion respectively, and the enzyme behind the purifying is cut product connect, connect product and transform DH-5 α competent cell, choose single white colony amplification, the extracting recombinant plasmid dna is made double digestion and is identified.

The a pair of primer sequence of above-mentioned DC-SIGN promotor is as follows:

Sense primer:ATCATACGCG TATGAGTCCT TCTCCCTGTC, contain Mlu I restriction enzyme site:

Antisense primer:TGTAAGATCT GTCACCCCAC TCTCCCCCAG contains Bgl II restriction enzyme site.

Description of drawings

Fig. 1 is the structure iron of pGL3-basic and pGL3-control.

Fig. 2 is DC-SIGN promotor amplification figure, and DNA marker is 100bp marker.

Fig. 3 is the sequence signature figure of DC-SIGN promotor.

Fig. 4 cuts figure as a result for the DC-SIGN-promoter-pGL3-Basic enzyme.The left side the 1st swimming lane is 200bp DNA ladder, the

2 swimming lanes are the contrast of DC-SIGN promoter PCR product, and the 3-7 swimming lane is DC-SIGN-promoter-pGL3-Basic double digestion result, and the 8th swimming lane is pGL3-Basic double digestion result, and the 9th swimming lane is λ/Hind III DNA ladder.

Fig. 5 is that the DC-SIGN promotor gets relative reactivity figure in obstructed cell.

Embodiment

1. make up the DC-SIGN promoter luciferase reporting plasmid

1.1 amplification DC-SIGN promotor

Get normal people's peripheral blood, separate PBMC, use the promotor of PCR primer amplification DC-SIGN gene.Used upstream primer is sense primer::ATC ATA CGC GTA TGA GTC CTT CTC CCT GTC, contain Mlu I restriction enzyme site, antisense primer:TGT AAG ATC TGT CAC CCC ACT CTC CCC CAG contains Bgl II restriction enzyme site.Use the Platinum_Taq DNA Polymerase High Fidelity of Invtrigon, the PCR reaction system is as follows:

5×PCRbuffer 2.5mM dNTP mix 50mM MgSO4 10mM sense primer 10mM antisense primer High Fidelity Taq(2U/μL) DNA(1ng/μL) DdH2O	10μL 4μL 2μL 2μL 2μL 0.5μL 1μL 28.5μL
		Total volumes	50μL

Increase on the PCR instrument by following program

1. 95℃×3min

2. 94℃×20sec

58℃×30sec

72 ℃ * 25sec is totally 30 circulations

3. 72℃×5min

44 ℃ of preservations

1.2 PCR product purification

Used the post centrifugation method, operation steps and method are carried out according to product description

1.3 make up the DC-SIGN promoter luciferase reporting plasmid

After the pGL3-basic plasmid increases through intestinal bacteria DH-5 α, use plasmid a small amount of extraction agent box to carry out extracting, the operation by specification carries out.Gained PCR is through Bgl II and Mlu double digestion with the gained plasmid with in 1.1 then, and used restriction endonuclease gets restriction endonuclease for Takara company, and system is as follows:

A. B

10×Buffer H 4μl 10×Buffer H 4μl

Bgl II(10U/μl) 1μl Bgl II(10U/μl) 1μl

Mlu(10U/μl) 1μl Mlu(10U/μl) 1μl

PGL3-Basic/Enhancer(0.1ug/μl) 10μl DC-SIGN promoter(50ng/μl) 20μl

ddH2O 24μl ddH2O 14μl

total volume 40μl total volume 40μl

With the light mixing of above-mentioned system, centrifugal, 37 ℃ of enzymes are cut and are spent the night.Enzyme is cut product and is carried out electrophoresis at 1% sepharose, and the electrophoretic voltage size is according to must be apart from setting between two electrodes of horizontal strip electrophoresis groove, and size is 5V/cm, after electrophoresis finishes, carries out dna gel and reclaims.Then the gained purifying being got DC-SIGN promoter fragment (40ng/ μ l) is connected with the T4 ligase enzyme with pGL3-Basic plasmid cleavage fragment (80ng/ μ l):

10×T4 ligation Buffer pGL3-Basic DC-SIGN promoter T4 ligase(5U/μl) ddH2O	1.0μl 3.0μl 1.0μl 1.5μl 3.5μl
		Total volume	10μl

With the said mixture mixing, centrifugal, 16 ℃ of reactions 3 hours.

Import competent cell 1.4 will connect product

Make and be divided in DH-5 α competent cell 100 μ l in the sterilization Ep pipe as on ice, operation as follows then with preparing solution with the single stage method competent cell in advance:

(1) competent cell precooling 20min on ice

(2) the connection product of adding 5 μ l in competent cell, mixing was placed 30 minutes on ice gently

Heat-shocked 90sec in (3) 42 ℃ of water baths placed 20 minutes immediately on ice

(4) add the nonresistant LB substratum of 800 μ l

(5) 37 ℃ of 200rpm shook 1 hour in horizontal constant temperature shaking table

(6) 4000rpm is centrifugal 5 minutes, removes 800 μ l supernatants

(7) with remaining 100 μ l liquid and precipitation mixing, be evenly coated on the LB solid culture flat board that contains penbritin (100 μ g/mL)

(8) earlier flat board is just being put 30min in 37 ℃ of bacteriological incubators, the bottom surface makes progress then, and bacterium faces down, 37 ℃ of overnight incubation.

(9) picking mono-clonal bacterium colony is put in 3mL and contains in the LB substratum of penbritin, vibrates 12-16 hour at 37 ℃ with 300rpm

(10) extract plasmid, carry out PCR and double digestion and identify

(11) will increase through identifying the LB that the bacterium liquid contain correct clone is put in 300mL (containing 100 μ g/mL)

(12) use the high-purity plasmid extraction test kit extracting DC-promoter-pGL3-Basic of amount in the plasmid

1.5 extracting DC-promoter-pGL3-Basic

(1) balance liquid EQ balance plasmid extraction Filter column

(2) with the centrifugal 5min of 300mL bacterium liquid 8000rpm, thoroughly remove supernatant

(3) add Lysis Buffer, thorough mixing, room temperature is placed 5min

(4) add among the neutralizer N buffer and 5min, 13000rpm high speed centrifugation 15min

(5) carefully get supernatant, add, filter, collect and filter gained liquid with balanced Filter column

(6) Virahol of adding 70% volume in the filter liquide of collecting, mixing

(7) the 13000rpm high speed centrifugation is 15 minutes, carefully removes supernatant

(8) 70% washing with alcohol precipitate once, and high speed centrifugation 10min carefully removes ethanol then

(9) will precipitate wind in the drying instrument to half-dried

(10) add ddH2O 400 μ L and melt plasmid

1.6 plasmid DC-SIGN-promoter-pGL3-Basic (DSPGB) transfecting eukaryotic cells

(1) transfection the day before yesterday, use to contain antibiotic culture medium culturing cell invariably, and by 0.6 * 104cell/ hole, inoculating cell is to 96 orifice plates

(2) plasmid is by 0.2 μ g/ hole (about 2 μ L), Opti-MEM reduced medium is by the standard in 25 μ L/ holes, with the ddH2O of 2 μ L as blank, 2 μ LpGL3-basic empty carriers are as negative control, 2 μ LDSPGB, with 2 μ LpGL3-control as positive control, be diluted among the Opti-MEM reduced medium mixing respectively

(3) Lipofectamine 2000 Transfection Reagent are diluted among the Opti-MEM of 25 μ L incubated at room 5min by the consumption in 0.5 μ L/ hole

(4) the Lipofectamine 2000 Transfection Reagent with dilution add among plasmid-Opti-MEM, mixing gently, incubated at room 20 minutes

(5) inhale the substratum that removes 96 well culture plate cells, add the Lipofectamine 2000-plasmid mixture book of 50 μ L, horizontal direction is shaken gently, makes the liquid mixture of adding be uniformly distributed in the culture plate bottom

Inhale after (6) 6 hours and remove aforesaid liquid, add ordinary culture medium

(7) hatched 30 hours at the cell cultures box column, detect the fluorescein activity then

1.7 uciferase activity detects

Detected uciferase activity in 30 hours after the transfection, carry out according to the following steps: (at room temperature carrying out)

(1) with Bright-Glo Luciferase buffer and Bright-Glo Luciferase substrate mixing, make Bright-GloLuciferase Assay reagent, packing, place-80 ℃ standby

(2) reagent among the Bright-Glo Luciferase Assay reagent melts from-80 ℃ of taking-ups, places 30min in room temperature

(3) cell is taken out from incubator, place 5min, inhale and go cells and supernatant in the culture plate,, remove PBS with PBS washing 1-2 time in room temperature

(4) add 100 μ LBright-Glo Luciferase Assay reagent,

(5) level vibration Glo Lysis Buffer guarantees that Bright-Glo Luciferase Assay reagent covers the diapire all cells several times

(6) incubated at room 5min

(7) detect at Orion II microplate luminometer, every hole detection time is 5sec

Interpretation of result:

1.DC-SIGN the amplification of promotor

The result of pcr amplification is carried out electrophoresis on 2% sepharose, the dna fragmentation size of gained is 237bp, as shown in Figure 1;

2.DC-SIGN the structure of promotor report carrier

Behind DC-SIGN promotor and pGL3-Basic double digestion, connect, behind the connection product transformed into escherichia coli, extract plasmid, carry out enzyme and cut evaluation, result such as Fig. 3;

3.DC-SIGN gene promoter active result's in obstructed cell mensuration

We find, the DC-SIGN promotor is relative higher with the activity in the Hela cell at Hacat, and active relatively low in HuVEC and Wish, this explanation, the DC-SIGN promotor is in different cells, different expression activities is arranged, and this expression activity may be relevant with intracellular microenvironment.Compare with the pGL3-control that contains the SV40 promotor, DC-SIGN promotor reporter gene produces to such an extent that uciferase activity is much lower.

Conclusion:

We have successfully made up the DC-SIGN promoter luciferase reporting plasmid, and it is imported respectively among Hela, HaCat, Wish and the HuVEC304, detect by uciferase activity, discovery is the expression activity difference in different cell strains, and expression activity is relative higher in the Hela cell strain of the HaCat in epidermic cell source and epithelial origin.

Sequence table

sense primer：ATCATACGCG TATGAGTCCT TCTCCCTGTC

antisense primer：TGTAAGATCT GTCACCCCAC TCTCCCCCAG

Claims (2)

1.DC-SIGN the construction process of promoter luciferase reporting plasmid is characterized in that following step:

(1) with reference to the DC-SIGN promoter sequence of GeneBank AF209479, the a pair of primer of design DC-SIGN promotor, introduce Mlu I and Bgl II recognition site respectively at 5 ' end and 3 ' end: ACGCGT and AGATCT, with PCR method high-fidelity amplification DC-SIGN promoter fragment, and purified pcr product, standby;

(2) DC-SIGN promoter fragment and its expression vector pGL3-basic are carried out double digestion respectively, and the enzyme behind the purifying is cut product connect, connect product and transform DH-5 α competent cell, choose single white colony amplification, the extracting recombinant plasmid dna is made double digestion and is identified.

2. according to the construction process of the described DC-SIGN promoter luciferase reporting plasmid of claim 1, it is characterized in that a pair of primer sequence of described DC-SIGN promotor is as follows:

sense primer：ATCATACGCG TATGAGTCCT TCTCCCTGTC

antisense primer：TGTAAGATCT GTCACCCCAC TCTCCCCCAG

Images