CN101591667A - Dectin-1 fusion protein expression vector and application - Google Patents

Abstract

The present invention relates to a kind of Dectin-1 fusion protein expression vector and application, also relate to structure by above-mentioned fusion protein expression vector, the present invention has successfully made up the Dectin-1 fusion protein expression vector, obtain the high Dectin-1 fusion rotein of purity behind this Dectin-1 fusion protein expression vector transfection recipient cell, and the solubility Dectin-1 fusion rotein that obtains can be fixed on the metallic surface of surface plasmon resonance biosensor, can monitor fungi fast, in real time, quantitatively, delicately.

Description

Dectin-1 fusion protein expression vector and application

Technical field

The present invention relates to a kind of structure of fusion protein expression vector, relate in particular to a kind of Dectin-1 fusion protein expression vector and application thereof.

Background technology

(nosocomial infections NI) is a great problem that current medical circle faces, and it not only has influence on the quality of medical treatment, and is concerning patient's safety in ward infection.At present, common especially in NI by the superinfection that the unreasonable use of antibacterials is caused, especially based on fungi infestation.Simultaneously, along with the widespread use clinically of microbiotic, immunosuppressor and corticosteroid hormone, generally the carrying out of organ transplantation, intubation catheter, the fungi infestation rate is more and more higher.Especially for immunosuppressant patient and critical patient, invasive infections with fungi becomes main infection complication already, and with very high morbidity and mortality ratio.

Because the early stage clinical symptom of various fungi infestations and sign are not special, the early diagnosis of fungi infestation depends on the detection to fungi.Chang Yong cultivation adds the morphology diagnostic method and still has low, the consuming time defective of a specified duration of susceptibility clinically.Although molecular biology method existing application the in the fungi diagnosis based on DNA detection is subjected to the obstruction of fungi cell wall, free fungal DNA limited amount in the sample makes positive rate low; The pre-treatment of sample has simultaneously also increased the possibility of fungal contamination, influences the accuracy of detected result.Some quick check methods based on fungal antigen and metabolite also progressively are applied to clinical.Wherein, with fungal cell wall polysaccharide component polygalactomannan and 1, the detection most worthy of 3-β-D dextran.Polygalactomannan is a kind of heat-stable mixed polysaccharide, be present in the cell walls of most of aspergillus tubigensis and Penicillium notatum, available ELISA method detects, but some Penicillium notatum deutero-microbiotic such as group draw XiLin, Ampicillin Trihydrate etc. can cause the intercrossing reaction, cause false-positive result.Often before all accept a large amount of antibiotic treatments because the patient of fungi infestation is taken place clinically, thereby influenced result's accuracy.Histopathology is particularly useful for deep fungal infection, but its operation has aggressive, and immunocompromise or patient with severe symptoms are also inapplicable.

Contain 1 on the cell walls of common fungi in the institute such as candidiasis, aspergillus tubigensis, (1,3-β-D-glucan, BG) composition, experiment in vitro show that the release of a large amount of BG will be arranged in the fungi growth phase to 3-β-D dextran ^[5]Interference experiment by multiple antibacterials simultaneously, find that these medicines are in reaching body under the condition of high Plasma Concentration, little (the Marty FM of influence that the mensuration of BG is caused, Lowry CM, Lempitski SJ, et al.Reactivity of (1,3)-β-D-glucan assay with commonly used intravenousantimicrobials.Antimicrob Agents Chemother.2006; 50:3450-3453), therefore, the detection of BG is used for diagnosing infection (Hope WW, Walsh TJ, the DenningDW.Laboratory diagnosis of invasive aspergillosis.Lancet Infect Dis 2005 of invasive fungi at present by the U.S. FDA approval; 5:609-622.).But, existing method for measuring be by BG can activation factor G the principle of (serine protease) realize that the factor G after the activation can activate proclotting enzyme in the king crab amoebocyte and change into and have active Thrombin coagulase; And the coagulagen in the amoebocyte is transformed into solidifying egg white, form gel (Hope WW, Walsh TJ, Denning DW.Laboratory diagnosis of invasive aspergillosis.Lancet Infect Dis 2005; 5:609-622.).This is similar to the bacterial endotoxin activation king crab approach that condenses, and therefore, contains the blue cloudy bacterium of endotoxic leather and can have considerable influence to this method, often needs pre-treatment to be removed, thereby makes that operation is more loaded down with trivial details.

Beta-glucan is the glucose polymer of one group of heterogeneity, for one of the abundantest polysaccharide of content in the fungal pathogens, accounts for 50% of fungal cell wall dry weight, is not only flexible but also firm necessary constituent of fungi cell wall.It mainly acts on is to activate granulocyte, has the ability of immunomodulator, and therefore a main target as fungal pathogens identification plays a significant role in the immunne response that starts host cell.

Host cell at the immunne response of beta-glucan mainly by the receptor protein mediation of cell surface.Known some cell surface receptors at present, as CR3, lactosylceramide, catch acceptor (scavenger receptor) and Dectin-1 receptor protein, they can both participate in cell recognition.Studies show that wherein Dectin-1 is bringing into play in cell response acts on.Dectin-1 is an II type transmembrane glycoprotein, molecular weight is 43KD, contain a single extracellular C type carbohydrate identified region (C-type carbohydrate recognition domain, CRD) and one intracytoplasmic based on tyrosine immunity receptor sample activation motif (immunoreceptor tyrosine-basedactivation motif, ITAM).Dectin-1 mainly is expressed in clones such as monocyte, scavenger cell and neutrophil leucocyte, and its expression amount obviously is subjected to the influence of cytokine and bacterial product.Dectin-1 can discern β-1 solubility or corpuscular, and 3-connects and β-1, and the dextran that 6-connects comprises complete fungal particles and the Unidentified part of T cell surface, thus the biological effect of mediation beta-glucan.

Summary of the invention

The object of the present invention is to provide a kind of Dectin-1 fusion protein expression vector and application thereof.

Dectin-1 fusion protein expression vector of the present invention comprises Dectin-1 target gene sequences and pSecTag2C carrier sequence.

Dectin-1 fusion protein expression vector of the present invention prepares by following method:

A. design and synthesize the PCR primer;

B. extract the RNA template;

C. carry out pcr amplification with RNA template among PCR primer and the b among the described a, obtain the Dectin-1 target gene sequences;

D. with the Dectin-1 goal gene that obtains among the c be connected after pSecTag2C carrier enzyme is cut, the transformed competence colibacillus cell;

E. from the described competent cell of d, extract recombinant plasmid and identify the Dectin-1 fusion protein expression vector that is obtained.

In the preferred embodiments of the present invention, the primer of PCR described in the above-mentioned steps a is the nucleotide sequence shown in the SEQ IDNO:1-2; The RNA template of the template of RNA described in the b for from Turnover of Mouse Peritoneal Macrophages, extracting.

Dectin-1 fusion rotein of the present invention is obtained by above-mentioned Dectin-1 fusion protein expression vector transfection recipient cell.

In the preferred embodiments of the present invention, above-mentioned recipient cell is 293 cells.

The invention still further relates to a kind of surface plasmon resonance biosensor, it is to be fixed with above-mentioned Dectin-1 fusion rotein at the sensor gold metal surface.

The invention still further relates to above-mentioned surface plasmon resonance biosensor or the application in detecting fungi.

The invention still further relates to the application of above-mentioned Dectin-1 fusion rotein in the preparation surface plasmon resonance biosensor.

The invention still further relates to the application of above-mentioned Dectin-1 fusion rotein in detecting fungi, in the preferred embodiments of the present invention, is above-mentioned ectin-1 fusion rotein to be fixed on utilize the ELISA method to detect fungi in the orifice plate.

The present invention has successfully made up the Dectin-1 fusion protein expression vector, obtain the high Dectin-1 fusion rotein of purity behind this Dectin-1 fusion protein expression vector transfection recipient cell, and the solubility Dectin-1 fusion rotein that obtains can be fixed on the metallic surface of surface plasmon resonance biosensor, can monitor fungi fast, in real time, quantitatively, delicately.

For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and conjunction with figs. are described in detail below.

Description of drawings

Fig. 1 is a Turnover of Mouse Peritoneal Macrophages extracting RNA electrophorogram;

Fig. 2 is the reverse transcription PCR electrophorogram, and marker is 50bp DNA Ladder Marker;

Fig. 3 A-3B is the grads PCR electrophorogram, and marker is 50bp DNALadder Marker;

Fig. 4 Dctin-1 gene PCR amplified production electrophorogram, marker is 50bp DNA LadderMarker;

Fig. 5 cuts the purified product electrophorogram for the enzyme of Dectin-1 gene PCR purified product and plasmid pSecTag2C, marker is 50bp DNA Ladder Marker, swimming lane 1 is a Dectin-1 gene PCR purified product, and swimming lane 2 is cut purified product for the enzyme of plasmid pSecTag2C;

Fig. 6 is a Dctin-1 gene PCR amplified production electrophorogram after the pSecTag2C-Dectin-1 recombinant plasmid transformed;

Fig. 7 is pSecTag2C-Dectin-1 recombinant plasmid (swimming lane 1) and empty carrier pSecTag2C (swimming lane 2) electrophorogram;

Fig. 8 is the gene sequencing result;

Fig. 9 is Dectin-1 fusion rotein western blot result, 1 is pSecTag2C plasmid transfection 293 cell culture medium purifying proteins, 2 is pSecTag2C-Dectin-1 rotaring redyeing 293 cell substratum purifying protein, and 3 is the 293 cell culture medium purifying proteins that do not deal with;

Figure 10 is fixed on the effect synoptic diagram that the method that can utilize ELISA in the orifice plate detects fungi for the Dectin-1 fusion rotein.

Embodiment

Embodiment 1The structure of Dectin-1 fusion protein expression vector

1, Turnover of Mouse Peritoneal Macrophages is extracted

(1) experimental article and instrument:

Kunming mice (do not limit by male and female, 22-25g, available from Medical University Of Chongqing's Experimental Animal Center), 6% starch meat soup (Zulkovsky starch 6g, peptone 1g, extractum carnis 0.3g, NaCl 0.5g, distilled water 100ml, high pressure steam sterilization), the 1ml syringe, 75% alcohol, cotton rod, beaker, Dissecting tray, Dissecting scissors, the ophthalmology tweezer, 20ml syringe (large size syringe needle), autoclaving PBS, sterilization 15ml centrifuge tube, DMEM (10% foetal calf serum, penicillin 100U/ml, Streptomycin sulphate 100ug/ml), the sterilization Tissue Culture Flask, spirit lamp, Bechtop, 37 ° of constant water bath box, 37 ° of CO ₂Incubator

(2) experiment content:

1. continuous two days to kunming mice abdominal injection 1ml sterilization back 6% starch meat soup

2. the 3rd day, kunming mice is taken off neck put to death, place 75% alcohol to soak 1 minute, take out mouse afterwards and be put in the Dissecting tray in the Bechtop, belly is upwards.

3. light spirit lamp, tweezers are mentioned the mouse abdomen near ventrimeson place skin, cut off an osculum, after upwards cut off skin of abdomen, expose peritonaeum fully.75% alcohol disinfecting peritonaeum.

4. open the PBS damping fluid in Bechtop, the 20ml syringe extracts 10mlPBS, punctures peritonaeum and injects mouse peritoneal, and to all directions flushing abdominal cavity, last pumpback peritoneal irrigation liquid injects the 15ml centrifuge tube several times in pumpback.

5. 500g, centrifugal 5 minutes, Turnover of Mouse Peritoneal Macrophages was at the pipe end.

6. with Bechtop in, discard supernatant liquor in the centrifuge tube, 3ml PBS is resuspended, the flushing cell, 500g, centrifugal 5 minutes.Repeat twice, with adherent starch on the flush away cell.

7. in Bechtop, discard supernatant liquor in the centrifuge tube, 3ml DMEM (10% foetal calf serum, penicillin 100U/ml, Streptomycin sulphate 100ug/ml) re-suspended cell sucks in the Tissue Culture Flask, is put in 37 ℃ CO ₂Incubator is cultivated.

8. after 4 hours, treat that Turnover of Mouse Peritoneal Macrophages is adherent after, in Bechtop, change liquid to cell.PBS flushing 2 times adds 3ml DMEM (10% foetal calf serum, penicillin 100U/ml, Streptomycin sulphate 100ug/ml), is put in 37 ℃ CO ₂Incubator is cultivated.

2, design of primers and synthetic

According to mouse Dectin-1 gene order (GenBank ID:AY534909) design primer, forward primer is SEQ ID NO:1-5 '-aaa GgtaccTagcattttggcgacac-3 ' contains restriction enzyme site KpnI (horizontal line mark part), and reverse primer is SEQ IDNO:2-5 '-aaa GaattcCcagttccttctcacagat-3 ' contains restriction enzyme site EcoRI (horizontal line mark part), and primer is synthetic by the handsome company in Shanghai.Goal gene is 534bp, expresses mouse Dectin-1 protein 67-244 amino acids.

3、RT-RCR

Experimental article and instrument: the total RNA extraction agent of UNIQ-10 pillar Trizol box (worker is given birth in Shanghai); RT-PCR reaction kit (TAKARA company); PrimerSTAR ^TMHS DNAPolymerase (TAKARA company); DEPC handles back autoclaving rifle head, PCR reaction tubes, 1.5mlEP pipe; The 1ml syringe; PCR instrument (U.S. BIO-RAD); Electrophoresis apparatus (Beijing Liuyi Instrument Factory); Gel imaging instrument (U.S. BIO-RAD); Eppendorf centrifuge (U.S. Thermo)

Experimental technique:

(1) RNA extracts (the total RNA extraction agent of UNIQ-10 pillar Trizol box)

1, with above-mentioned Turnover of Mouse Peritoneal Macrophages, its substratum is with PBS flushing 2 times.The Trizol that in culturing bottle, adds 0.5ml.

2, use the 1ml syringe, twice of 26-G syringe needle suction homogenate night to be to shear genomic dna, then directly from syringe with sample transfer in aseptic 1.5mlEP pipe.

3, add 100ul chloroform/Virahol (24: 1), thermal agitation mixing 30 seconds.

4, on the desk centrifuge, 12000rpm, centrifugal 5 minutes of room temperature.

5, supernatant liquor (about 450ul) is carefully transferred in the aseptic 1.5ml RNase-free centrifuge tube, added the 150ul dehydrated alcohol, mixing.

6, above-mentioned solution is all transferred to cover and be put in the UNIQ-10 post in the 2ml collection tube, room temperature was placed 2 minutes, centrifugal 1 minute of 8000rpm.

7, carefully take out pillar, discard the waste liquid in the collection tube, pillar is put back in the collection tube, add 10ul DNase in the UNIQ-10 post, room temperature left standstill 10 minutes, centrifugal 1 minute of 8000rpm.

8, carefully take out pillar, discard the waste liquid in the collection tube, pillar is put back in the collection tube, add 450ul RPE Solution, 10000rpm, centrifugal 30 seconds of room temperature.

9, repeating step 8

10, carefully take out pillar, discard the waste liquid in the collection tube, pillar is put back in the collection tube, 10000rpm, centrifugal 15 seconds of room temperature.

11, carefully take out pillar, put into the 1.5ml centrifuge tube of aseptic 1.5ml RNase-free, at the careful 50ulDEPC-H that adds of the central authorities of post inner membrance ₂O placed 2 minutes for 55-80 ℃.

12,10000rpm, centrifugal 1 minute of room temperature.Solution in the collection tube is the RNA sample, can use immediately or-70 ℃ of preservations.

13, ultraviolet spectrophotometer is surveyed RNA sample concentration and purity.The RNA sample concentration is 25ug/ml, OD _260/280＞1.8

14, electrophoresis is identified the RNA sample purity.(see figure 1).

(2) RT (reverse transcription)

Reaction conditions: 70 ℃ 10 minutes, 0 ℃ 4 minutes

Centrifugal 10 seconds

Reaction conditions: 42 ℃ 1 hour, 70 ℃ 15 minutes, 0 ℃ of 10 minutes final cDNA product 100ul ,-20 ℃ of preservations

(3)PCR

①PCR：

Reaction system:

Reaction conditions:

Electrophoresis is identified: (see figure 2).

2. grads PCR

Reaction system:

Reaction conditions:

Electrophoresis is identified: annealing temperature should be 62 ℃.(seeing Fig. 3 A-3B).

③PCR

Reaction system:

Reaction conditions:

Electrophoresis is identified: PCR product-20 ℃ preservation.(see figure 4).

4, construction of recombinant plasmid and evaluation

Experiment material and instrument:

E.Z..N.A. ^TMCycle-Pure Kit (Omega company), E.Z..N.A. ^TMPlasmid MiniKit (Omega company), T4DNA ligase

Experimental technique:

(1) above-mentioned PCR product purification (E.Z..N.A. ^TMCycle-Pure Kit):

1, balance pillar: get HiBand DNA post and be put in and 1. add 200ulGPS in the 2ml collection tube, room temperature left standstill 3-5 minute, 12000g, and 2. 2 minutes centrifugal abandons collection tube filtrate, adds 700ul ddH in HiBand DNA post ₂O, 12000g, 3. 2 minutes centrifugal abandons collection tube filtrate, and pillar is put back in the collection tube, and promptly balance is good.The HiBand DNA Column that balance is good can place 1-2 week at room temperature.

2, the PCR product of collecting among above-mentioned 3 (the 3. PCR) is moved in the aseptic 1.5ml reaction tubes, add the CP damping fluid of 4-5 times of volume, add the Virahol of cumulative volume 1/3 again, mixing.(, then adding the Buffer CP of 6 times of volumes) as PCR product＜500bp

3, above-mentioned solution is added in the good HiBand DNA post of balance, room temperature, centrifugal, 10000g, 1 minute.

4, abandon collection tube filtrate, reuse collection tube, as mixeding liquid volume＞700ul in 2, then repeating step 3.

5, add the 700ulDNA elution buffer in HiBand DNA post, room temperature, centrifugal, 10000g, 1 minute.

6, abandon collection tube filtrate, repeating step 5.

7, abandon collection tube filtrate, the HiBand DNA post of centrifugal sky, room temperature, 13000g, 2 minutes.

8, HiBand DNA post is put in the aseptic 1.5mlEp pipe, adds the 30ul elution buffer, leave standstill 1-2 minute under the room temperature, centrifugal, 13000g, 1 minute.

9, ultraviolet spectrophotometer is measured PCR purified product concentration: 107ug/ml ,-20 ℃ of preservations.

(2) PCR purified product and pSecTag2C plasmid double digestion:

Reaction system:

Reaction conditions: enzyme Qie Wendu: 37 ℃

Enzyme is cut the time: purpose fragment: 5-6 hour; Plasmid: about 8 hours

(3) enzyme is cut product purification (E.Z..N.A. ^TMCycle-Pure Kit):

1, balance pillar: get HiBand DNA post and be put in and 1. add 200ul GPS in the 2ml collection tube, room temperature left standstill 3-5 minute, 12000g, and 2. 2 minutes centrifugal abandons collection tube filtrate, adds 700ul ddH in HiBand DNA post ₂O, 12000g, 3. 2 minutes centrifugal abandons collection tube filtrate, and pillar is put back in the collection tube, and promptly balance is good.The HiBand DNA post that balance is good can be placed 1-2 week at room temperature.

2, the enzyme of PCR purified product in above-mentioned (2) and plasmid pSecTag2C is cut product and moved to respectively in the aseptic 1.5mlEp pipe, add the CP damping fluid of 4-5 times of volume, add the Virahol of cumulative volume 1/3 again, mixing.(, then adding the CP damping fluid of 6 times of volumes) as PCR product＜500bp.

3, above-mentioned solution is added in the good HiBand DNA post of balance, room temperature, centrifugal, 10000g, 1 minute.

4, abandon collection tube filtrate, reuse collection tube, as mixeding liquid volume＞700ul in 2, then repeating step 3.

5, add the 700ulDNA elution buffer in HiBand DNA post, room temperature, centrifugal, 10000g, 1 minute.

6, abandon collection tube filtrate, repeating step 5.

7, abandon collection tube filtrate, the HiBand DNA Column of centrifugal sky, room temperature, 13000g, 2 minutes.

8, HiBand DNA Column is put in the aseptic 1.5mlEp pipe, adds the 30ul elution buffer, leave standstill 1-2 minute under the room temperature, centrifugal, 13000g, 1 minute.

9, the enzyme that promptly obtains PCR purified product and plasmid pSecTag2C is cut purified product.

10, the enzyme of PCR purified product and plasmid pSecTag2C is cut purified product and carry out agarose gel electrophoresis, with 50bp marker is contrast, the enzyme of seeing PCR purified product and plasmid pSecTag2C is cut the brightness and the 50bp marker contrast of the electrophoretic band of purified product, the concentration that the enzyme of reckoning PCR purified product and plasmid pSecTag2C is cut purified product has been carried out ligation.(see figure 5).

(4) connect:

Reaction system:

Reaction conditions:

45 ° were heated 5 minutes, and were cooled to 0 ℃ afterwards

16 ℃ spend the night (about 8h), 72 ℃ of deactivations 10 minutes.Can obtain recombinant plasmid ,-20 ℃ of preservations.

(5) transform:

1. prepare competent cell:

1, get DH5 α bacterium liquid 5ul and be inoculated in the 5mlLB liquid, 37 ℃ of shaking culture are spent the night

2, second day with bacteria suspension with (4 pipe) in the LB substratum that is inoculated in 100ml at 1: 50,37 ℃ of shaking culture, after general 1.5 hours, visible cloud is vaporific, stops to cultivate.

3, place 10 minutes on ice.

4,4 ℃ of centrifugal 3000g, 10 minutes.

5, abandon supernatant, add the CaCl of isopyknic ice-cold 0.1M ₂Solution, vibration gently, suspension cell.

6, place 30 minutes on ice

7,4 ℃ centrifugal, 3000g, 10 minutes.Abandon supernatant, add the CaCl of the ice-cold 0.1M of 4ml (containing 9%DMSO) ₂Solution.Suspension cell was placed 5 minutes on ice gently.

8, above-mentioned suspension branch is installed in the EP pipe of 1.5ml every pipe 100ul.In placing and be put in 4 ℃ of refrigerators on ice 1-2 hour, quick-frozen in liquid nitrogen can be used immediately, or-80 ℃ of preservations.

2. transform

1, the recombinant plasmid 2ul that gets above-mentioned structure joins in the competent cell of 200ul, shakes up gently.

2, place 30 minutes on ice.

3,42 ℃ of water-bath thermal shock 90-120 seconds

4, rapid cooled on ice is 2 minutes

5, in above-mentioned Ep pipe, add 1ml LB liquid nutrient medium immediately, after shaking up, 37 ℃, shaking culture, 1 hour

6, get the 100ul aforesaid liquid, on inoculation and the LA plate (100ug/ml), smoothen with glass stick.

7, culture plate is put in 37 ℃ of constant incubators, treat that bacterium liquid absorbs fully after 2 hours after, be inverted incubated overnight

8, do the positive, negative control simultaneously:

Positive control: do not transform plasmid, be coated with the LB plate, should grow bacterium +++

Negative control: do not transform plasmid, be coated with the LA plate, should not grow bacterium-

Other: transform empty plasmid (pSecTag2C), be coated with the LA plate, should grow bacterium ++

(6) bacterium colony PCR:

The preparation template:

Get 19 aseptic PCR reaction tubess,, respectively add 10ulddH from 1 mark to 19 ₂O, then, 18 single bacterium colonies of picking on the LA plate after the above-mentioned recombinant plasmid transformed are added in this 1-18 number aseptic PCR reaction tubes mixing respectively; 1 single bacterium colony of picking on the LA plate after transforming from empty plasmid (pSecTag2C) is added in No. 19 aseptic PCR reaction tubess mixing.

Reaction system:

Outside the removing template (1ul), with all the other solution be added in 1 aseptic 1.5mlEP pipe, mixing, its branch is installed in 19 PCR reaction tubess, 9ul/ pipe mark 1-19 number, adds template 1ul respectively.Be that each reaction system is 10ul.

Reaction conditions:

3% agarose gel electrophoresis: (see figure 6).

(7) extract recombinant plasmid (E.Z..N.A. ^TMPlasmid Mini Kit):

1, according to electrophorogram, get 2,3,7,8,9,10,12, No. 17 bacterium liquid and add in the 2ml LA liquid, preserving bacterial classification, No. 18 bacterium liquid add to 6ml LA liquid (Ampicillin Trihydrate, 100ug/ml) in, to preserve bacterial classification and extracting plasmid.37 ℃, 200rpm, shaking culture is spent the night.

2, balance pillar: get HiBind in a small amount preparative column be put in and 1. add 200ulGPS in the 2ml collection tube, room temperature left standstill 3-5 minute, 12000g, 2. 2 minutes centrifugal abandons collection tube filtrate, adds 700ulddH in the preparative column in a small amount to HiBind ₂O, 12000g, 3. 2 minutes centrifugal abandons collection tube filtrate, and pillar is put back in the collection tube, and promptly balance is good.The HiBind a small amount of preparative column that balance is good can be placed 1-2 week at room temperature.

3, No. 18 bacterium liquid 1.5-5ml that shaken are sub-packed in some aseptic 1.5mlEp pipes 10000g, centrifugal 1 minute.

4, abandon supernatant liquor, in the Ep pipe, add 250ul Solution I/Rnase A, resuspended bacterium.

5, add 250ul Solution II, and mixing gently, up to obtaining a clarifying lysate.Room temperature left standstill 2 minutes.

6, add 350ul Solution III, mixing immediately is up to white insolubles occurring.

7, room temperature is centrifugal 10 minutes, 〉=10000g.

8, carefully draw about supernatant liquor 700ul, add in the HiBind a small amount of preparative column centrifugal 1 minute of room temperature 10000g.

9, abandon filtrate, reuse the 2ml collection tube, add the HB damping fluid flushing HiBind a small amount of preparative column of 500ul, centrifugal 1 minute of room temperature, 10000g.

10, abandon filtrate, reuse the 2ml collection tube, add 700ulDNA dcq buffer liquid (having added ethanol) in the preparative column in a small amount to HiBind.Centrifugal 1 minute of room temperature, 10000g.

11, repeating step 10.

12, abandon filtrate, reuse the 2ml collection tube, centrifugal void column 2 minutes, 〉=13000g.

13, HiBind a small amount of preparative column is put in the aseptic 1.5ml Ep pipe, adds the 30-50ul elution buffer, room temperature left standstill 1-2 minute, and centrifugal 1 minute, 13000g.Can obtain plasmid solution.

14, ultraviolet spectrophotometer is measured plasmid concentration.

15,1% agarose gel electrophoresis.(see figure 7).

5, dna sequence dna order-checking

With above-mentioned plasmid, get 10ul and send with Medical University Of Chongqing and check order.The target gene sequences of the Dectin-1 fusion rotein carrier for expression of eukaryon that makes up is 759bp, the sequencing result result conforms to substantially with pSecTag2C plasmid, GenBank ID:AY534909, except that the 161st, 538 base, but also conform to GenBank ID:AF262985.(see figure 8).

Embodiment 2The Dectin-1 expressing fusion protein

Recombinant plasmid (pSecTag2C-Dectin-1) contains Igk protein excretion expressing gene, to guarantee protein expression; Contain 6 * His label and be beneficial to protein purification; Be not with the green fluorescent protein mark.

Experiment material: the DMEM of antibiotic-free serum-free, 10% foetal calf serum antibiotic-free DMEM, liposome 2000,

Ni-NTA?His-Bind?Resin

(1) transfection:

1, transfection the day before yesterday, inoculate 293 cells to three, 6 orifice plate, transfection same day, cell will reach 90%～95% degree of converging (substratum is the DMEM of antibiotic-free)

2, added before liposome/DNA mixture in the short period of time transfection same day, sops up former substratum, with the PBS flushing once, changes the DMEM substratum of 1ml antibiotic-free serum-free

3, prepare following mixture:

(1) dilutes recombinant plasmid (4ug) in the DMEM of 250ul serum-free antibiotic-free, gently mixing.Incubated at room 5 minutes.

(2) use liposome 2000 preceding first mixings, draw the DMEM that an amount of (10ul) is diluted to 250ul serum-free antibiotic-free.Incubated at room 5 minutes.

(3) incubated at room is after 5 minutes, the recombinant plasmid of mixed diluting and the liposome of dilution 2000 (500ul volume altogether), mixing gently, 20 minutes (cloud may appear in solution) of incubated at room.(mixture can at room temperature be stablized 6 hours)

4, dropwise above-mentioned 500ul mixture is added in 293 cells, 6 orifice plates in the hole (not removing substratum) limit edged jog culture plate

5,37 ℃, CO ₂Incubator was hatched 18-48 hour, hatched to change 10% foetal calf serum antibiotic-free DMEM after 4-6 hour.

6, transfection recombinant plasmid 5 holes; The empty plasmid of transfection simultaneously pSecTag2C to 293 cell, 5 holes; Do not deal with 293 cells, 5 holes; Transfection is with reference to plasmid PEGFP-N ₁(being marked with green fluorescent protein on the plasmid) 1 hole.

7, after the transfection 24 hours, see under the inverted fluorescence microscope with reference to plasmid PEGFP-N again ₁Transfection efficiency, transfection efficiency can reach 60%-70%.

(2) collect sample:

Collect after recombinant plasmid, the empty plasmid pSecTag2C transfection 24 hours, 48 hours, the 72 hours substratum and 293 cells that are untreated with the time point substratum ,-70 ℃ of preservations.

(3) concentrated, the purifying of albumen:

1, reagent preparation:

1 * bag is equipped with damping fluid: 50mM NaH ₂PO ₄, pH 8.0; 300mM NaCl; 10mM imidazoles (imidazole)

1 * dcq buffer liquid: 50mM NaH ₂PO ₄, pH 8.0; 300mM NaCl; 20mM imidazoles (imidazole)

1 * elution buffer: 50mM NaH ₂PO ₄, pH 8.0; 300mM NaCl; 250mM imidazoles (imidazole)

2, albumen concentrates:

With the 10KD ultrafiltration pipe protein concentrate sample of 15ml, it is red to be equipped with damping fluid (BindBuffer) dephenolize with above-mentioned bag then.That is, sample is equipped with in the 10KD ultrafiltration pipe that adds to 15ml after the damping fluid dilution 4 ℃ with bag, 3000g, centrifugal 15min, several times repeatedly, to protein sample concentrate, dephenolize is red.

3, protein purification:

1. be equipped with damping fluid balance pillar with bag.

2. add sample to post, collect filtrate western blot and identify.

3. use 2 * 4ml dcq buffer liquid (Wash Buffer) to wash pillar, collect elutriant western blot and identify.

4. use 4 * 1ml elution buffer (Elute Buffer) soluble protein, collect purifying protein western blot and identify.

5. western blot detects

Embodiment 3Dectin-1 fusion rotein western blot identifies

(1) join glue:

After adding separation gel, it is with fixed attention short to add a little water.When treating between water and the glue to form a broken line then glue coagulate; Sucking-off moisture also blots with thieving paper, adds concentrated glue, fills it up with, in order to avoid produce bubble as far as possible; After treating gelling, take out comb, wash each hole, get final product with electrophoresis liquid.

(2) go up sample: albumen (16ul)+water (0ul)+5 * sample-loading buffer (4ul), 98 ℃ 10 minutes, sample is gone up in centrifugal back.Add protein marker (8ul).

(3) electrophoresis: the inside groove electrophoresis liquid is filled it up with, and water jacket is added to the minimum rate of accumulation, first voltage 80V electrophoresis 15-20 minute, and the 120V electrophoresis is about 1.5 hours again.

(4) change film: film liquid is changeed in precooling.PAGE glue corner cut makes marks, (first corner cut makes marks with pvdf membrane then, soak with methyl alcohol), filter paper, PAGE glue are cut into suitable big vesicle electricity changeed liquid 15 minutes, taking out by black flour is that the order of the end+cellulose plate+filter paper+glue+film+filter paper+cellulose plate+fine flour installs.Black flour is put into electric rotary device to black flour.Be placed in the ice chest 100V constant voltage and changeed film 1 hour.

(5) sealing: take out film and put in the confining liquid room temperature sealing 2 hours.

(6) one is anti-: diluted anti-His antibody than row with confining liquid by 1: 1000, adding is put the film plastic tape and is sealed, 4 ℃ of overnight incubation.Washed 3 * 10 minutes with PBST then.

(7) two is anti-: washed film is put into plastic tape, and seal two anti-(1: 500) that add the confining liquid dilution, puts and shook in the shaking table under the room temperature 1-2 hour, washed 3 * 10 minutes with PBST then.

(8) DAB colour developing: add one of A liquid, mixing among the 1-1.5ml; Add each one of B and C liquid then, mixing.Lucifuge was used in 30 minutes.

(9) see Fig. 9.

Embodiment 4The preparation of surface plasmon resonance biosensor

Fixedly one deck acceptor molecule is the Dectin-1 fusion rotein of above-mentioned purifying in the metallic surface, and the part and the reaction between the fixed acceptor that are added in the damping fluid will cause SPR spectrum peak that the displacement that can observe takes place.These information data transmission are to computer, by analyzing the interaction that can monitor in real time, quantitatively, delicately between biomacromolecule.By the solubility Dectin-1 fusion rotein of purifying being fixed on the metallic surface of surface plasmon resonance biosensor, can monitor fungi fast, in real time, quantitatively, delicately.

The present invention can also be fixed in the orifice plate by the solubility Dectin-1 fusion rotein with purifying and can utilize the method for ELISA to detect fungi, as shown in figure 10, and wherein

The expression orifice plate

The solubility Dectin-1 fusion rotein of expression purifying

Biotinylated solubility Dectin-1 fusion rotein

The expression fungi

Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when can doing a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.

Sequence table

＜110〉the three or three affiliated hospital of Military Medical Univ No.3, P.L.A, No.1 Hospital Attached to the Chongqing Medical University

＜120〉Dectin-1 fusion protein expression vector and application

<130>

<160>2

<170>PatentIn?version?3.3

<210>1

<211>26

<212>DNA

＜213〉primer sequence of amplification Dectin-1 gene order

<400>1

aaaggtacct?agcattttgg?cgacac 26

<210>2

<211>28

<212>DNA

＜213〉primer sequence of amplification Dectin-1 gene order

<400>2

aaagaattcc?cagttccttc?tcacagat 28

Claims (10)

1. a Dectin-1 fusion protein expression vector is characterized in that comprising Dectin-1 target gene sequences and pSecTag2C carrier sequence.

2. the preparation method of the described Dectin-1 fusion protein expression vector of claim 1 is characterized in that comprising step:

A. design and synthesize the PCR primer;

B. extract the RNA template;

C. carry out pcr amplification with RNA template among PCR primer and the b among the described a, obtain the Dectin-1 target gene sequences;

D. with the Dectin-1 goal gene that obtains among the c be connected after pSecTag2C carrier enzyme is cut, the transformed competence colibacillus cell;

E. from the described competent cell of d, extract recombinant plasmid and identify the Dectin-1 fusion protein expression vector that is obtained.

3. method according to claim 2 is characterized in that the primer of PCR described in a is the nucleotide sequence shown in the SEQID NO:1-2.

4. method according to claim 2 is characterized in that the RNA template of the template of RNA described in the b for extracting from Turnover of Mouse Peritoneal Macrophages.

5. a Dectin-1 fusion rotein is characterized in that being obtained by the described Dectin-1 fusion protein expression vector of claim 1 transfection recipient cell.

6. Dectin-1 fusion rotein according to claim 5 is characterized in that described recipient cell is 293 cells.

7. a surface plasmon resonance biosensor is characterized in that being fixed with claim 5 or 6 described Dectin-1 fusion roteins at the sensor gold metal surface.

8. the application of the described surface plasmon resonance biosensor of claim 7 in detecting fungi.

9. claim 5 or the 6 described Dectin-1 fusion roteins application in the preparation surface plasmon resonance biosensor.

10. claim 5 or the 6 described Dectin-1 fusion roteins application in detecting fungi.

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