CN101628942A - Recombinant fusion protein based on human TMPD1 and HMGB1 A box and preparation method thereof - Google Patents

Abstract

The invention provides a recombinant fusion protein based on human TMPD1 and HMGB1 A box, which has an amino acid sequence shown in SEQ ID NO:1 and a nucleotide sequence shown in SEQ ID NO:2. The invention also provides a preparation method of the recombinant fusion protein. After the recombinant fusion protein is obtained by a gene engineering strategy, the detection of the in-vitro inflammation-resisting action of the recombinant fusion protein shows that the recombinant fusion protein can effectively suppress the release of TNF-alpha induced by human HMGB1 with a definite inflammation causing action, and the suppressing action is obviously better than that of independent HMGB1 A box and independent TMPD1.

Description

Based on recombination fusion protein of people TMPD1 and HMGB1 A box and preparation method thereof

Technical field

The present invention relates to biotechnology, bio-pharmaceutical field, particularly, the present invention relates to high mobility group protein B 1 (High-mobility group box-1, HMGB1) the anti-inflammatory fusion molecule that PD1 (TMPD1) structural domain in A box structural domain in (HMGB1 A box) and the thrombomodulin (Thrombomodulin TM) is formed, and preparation method thereof.

Background technology

The septicemia that infection causes causes multiple organ dysfunction syndrome, and mortality ratio is up to 30%～50%, and active and effective anti-inflammatory measure is the prerequisite that reduces mortality ratio.HMGB1 is a kind of important pro-inflammatory cytokine of finding in recent years.After it is released to outside the born of the same parents, just can bring into play its extremely strong proinflammatory effect, thereby it plays crucial effects in local and systemic inflammatory and the syndromic generating process of toxicity by a large amount of releases that stimulate multiple pro-inflammatory cytokine more enduringly in born of the same parents.

At present, HMGB1 has as the material evidence of crucial pro-inflammatory cytokine: (1) HMGB1 and B box thereof can stimulate Monocytes, neutrophilic granulocyte to discharge multiple pro-inflammatory cytokines such as TNF α, the HMGB1 of extremely low concentration gets final product activated macrophage, and prompting HMGB1 may be that TNF α discharges the most important endogenous stimulus factor.(2) the HMGB1 wide expression is in various kinds of cell.Therefore, can discharge the HMGB1 except infecting the activated immunocyte, other the impaired or downright bad somatocyte that causes because of factors such as ischemic, wound, burns also can discharge HMGB1 in a large number, and (existing experiment shows, at the HMGB1 knock-out mice, its cell lysate reduces greatly than the scorching ability of causing of wild-type cell lysate), the wide material sources of visible HMGB1.The popularity in its this source, with infect and inflammatory reaction integration that non-infectious factor causes together, thereby to a great extent reasonable dismissal infect the systemic inflammation that causes with non-infectious mechanism why and finally all develop into the similar toxicity syndrome of clinical symptom.(3) handle and dead mouse through HMGB1, its histopathological examination finds seldom have the tissue of vitals to necrose, and this is consistent with the postmortem result who much dies from the syndromic patient of toxicity.Its mechanism may be that HMGB1 makes epithelial cell can not keep tight connection, thereby the epithelium dysfunction that has caused popularity, the result makes body fluid and ionogen be shifted, and can't keep the balance of intercellular body fluid and ionogen and energy gradient, finally causes body death.(4) experimentation on animals shows, the inhibitor of HMGB1 has good preventive and therapeutic effect to systemic inflammatory and toxicity syndrome, can significantly increase the animal dis motility rate; Even give the inhibitor of HMGB1 after 24 hours in morbidity, still can bring into play therapeutic action effectively, that is to say that the treatment window phase at HMGB1 can reach 〉=24 hours.As seen, the anti-inflammatory measure operability clinically at HMGB1 is stronger.

In recent years studies show that (The Journal of Clinical Investigation, 2005 115:1267-1274), TM can produce direct, tangible anti-inflammatory effect by the effect of the combination of PD1 structural domain and the crucial pro-inflammatory cytokine HMGB1 that neutralizes.Discoveries such as Kazuhiro, TMPD1 has clear and definite anti-inflammatory properties, and this structural domain combines HMGB1 B box with the HMGB1 receptor competition, in experiment in vitro, stop leukocytic activation, suppress NF-κ B signal path, suppress the release of TNF α, inflammatory factors such as IL-1, IL-8; The experimentation on animals that mouse carries out is found that TMPD1 can obviously be alleviated UV-induced skin inflammation, and reduce the mortality ratio that LPS causes.

Summary of the invention

In view of HMGB1 A box is a small peptide that has only 85 amino-acid residues to form, property that expression in bacterium is understable, and TMPD1 also has these characteristics, the present invention is intended to TMPD1 gene and HMGB1A box encoding sequence are merged, with obtain to have double inhibition HMGB1's and the better fusion rotein of stability, this fusion rotein under its effect, can be realized the efficient blocking-up to the HMGB1 signal path as the new inhibitor of HMGB1.

Recombination fusion protein based on people TMPD1 and HMGB1 A box of the present invention has the aminoacid sequence shown in the SEQID NO:1; Has the nucleotide sequence shown in the SEQ ID NO:2.

The present invention also provides the preparation method of above-mentioned recombination fusion protein based on people TMPD1 and HMGB1 A box, mainly comprises following steps:

1) extracts total RNA of human peripheral blood mononuclear cell and be cDNA with its reverse transcription;

2) be template with the cDNA in the step 1), use the nucleotides sequence shown in the SEQ ID NO:3-4 to classify the cDNA of primer amplification people TMPD1 as;

3) be template with the cDNA in the step 1), use the nucleotides sequence shown in the SEQ ID NO:5-6 to classify the cDNA of primer amplification people HMGB1 A box as;

4) with step 2) cDNA of the people TMPD1 that obtains and the cDNA of the people HMGB1 A box that step 3) obtains respectively enzyme cut the back and be connected with carrier;

5) the step 4) gained is connected the product transformed into escherichia coli, screening positive clone must comprise the recombinant vectors of people HMGB1A box/ restriction endonuclease sites/TMPD1;

6) be template with the step 5) recombinant vectors,, in disappearance restriction endonuclease sites encoding sequence, people HMGB1 A box directly linked to each other with the encoding sequence of TMPD1 through inverse PCR;

7) the PCR product transformed into escherichia coli that directly links to each other of the people HMGB1 A box that step 6) is obtained and TMPD1, the recombinant protein that the abduction delivering acquisition is fused to TMPD1 by HMGB1 A box.

Wherein, use KpnI and EcoRI enzyme to cut step 2 in the step 4)) cDNA of the people TMPD1 that obtains; Use EcoRI and HindIII enzyme to cut step 2) cDNA of the people HMGB1 A box that obtains.

Wherein, the restriction endonuclease sites in the step 5) is preferably EcoR I.

Recombination fusion protein based on people TMPD1 and HMGB1 A box of the present invention can be used to prepare anti-inflammatory drug.

Behind the recombination fusion protein of the present invention that gene engineering strategy obtained, it is carried out the extracorporeal anti-inflammatory effect and detect demonstration, can effectively suppress to have the release of the people HMGB1 inductive TNF-α of clear and definite proinflammatory effect, and this restraining effect obviously is better than independent people HMGB1 A box and TMPD1.

The present invention by engineered construction of strategy by HMGB1 A box that all has the obvious anti-inflammatory and anti effect and fusion molecule that TMPD1 forms, its advantage is: (1) existing verified independent HMGB1 A box of research and TMPD1 all has significantly, different mechanisms is at the anti-inflammatory action of this individual interior crucial pro-inflammatory cytokine of HMGB1, therefore, the two is merged, to obtain to have the novel derived molecules of double inhibition HMGB1; (2) HMGB1 A box is a small peptide that has only 85 amino-acid residues to form, property that the expression in bacterium is understable, and be not easy to purifying, and by merging, increase its molecular weight, stablize the good method of its expression beyond doubt; (3) TMPD1 is clear and definite and single at the restraining effect of inflammation system, so side effect is little, is one of optimal selection as HMGB1A box fusion partner; (4) pQE 80L/DH5 α system is the expression system that the inventor optimizes out by screening, and it has plurality of advantages, and obtains to have the target protein of 6 * His labelled peptide, is easy to purifying, has improved the productive rate of fusion rotein greatly.

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

The behave schematic diagram of TMPD1/HMGB1 A box gene clone of Fig. 1;

Fig. 2 is the collection of illustrative plates of pUC19 carrier;

Fig. 3 is the step synoptic diagram of a step inverse PCR;

Fig. 4 is the dna sequencing collection of illustrative plates;

Fig. 5 is a pQE 80L plasmid map;

Fig. 6 is the electrophorogram of reorganization prokaryotic expression plasmid pQE80L-TMPD1/HMGB1 A box after enzyme is cut, and wherein 1:DNA Maker 2000; 2:pQE80L-TMPD1/HMGB1 A box KpnI/HindIII double digestion;

Fig. 7 is protein immunoblotting collection of illustrative plates, wherein A:1: low molecular weight protein (LMWP) marker; 2:DH5 α/pQE80L reorganization bacterium IPTG induces the back expression product; 3:DH5 α/pQE80L-TMPD1/HMGB1 A box reorganization bacterium IPTG induces the back expression product; Inclusion body after 4:DH5 α/pQE80L-TMPD1/HMGB1 A box reorganization bacterium IPTG induces; B:1:DH5 α/pQE80L-TMPD1/HMGB1 A box reorganization bacterium IPTG induces the back expression product to analyze through Western blot;

Fig. 8 is SDS-PAGE electrophoresis result 1: low molecular weight protein (LMWP) marker; 2: the fusion rotein TMPD1/HMGB1 A box behind the purifying;

Fig. 9 suppresses the result that HMGB1 induces monocyte TNF secretion-α for detect reorganization TMPD1/HMGB1 A box with ELISA.

Embodiment

Total technical scheme of the preparation method of recombination fusion protein of the present invention:

(1) with monocyte separation medium separation of human peripheral blood lymphocytes in whole blood, proposing total RNA and reverse transcription is cDNA.

(2) be template with (1), through the cDNA of conventional pcr amplification coding people TMPD1, the end upstream and downstream primer sequence that is used is respectively: 5 '-GTT GGTACCGAGCCGCAGCCGGGTGGCAG-3 '; (restriction endonuclease sites of base shown in the underscore for introducing, KpnI) 5 ' GCG GAATTCCCTGCAGGTGGCTGGGAAGTGGA-3 ', (restriction endonuclease sites of base shown in the underscore for introducing, EcoRI).After stating two kinds of restriction enzymes double zyme cutting people TMPD1 cDNA fragments in the use, reclaim the purpose fragment.

(3) be template with (1), through the cDNA of conventional pcr amplification coding people HMGB1 A box, the upstream and downstream primer sequence that is used is respectively: 5 '-GTT GAATTCATGGGCAAAGGAGATCCTA-3 ' (restriction endonuclease sites of base shown in the underscore for introducing, EcoRI); 5 '-GCG AAGCTTTGTCTCCCCTTTGGGA-3 ' (restriction endonuclease sites of base shown in the underscore for introducing, HindIII).After stating two kinds of restriction enzymes double zyme cutting people HMGB1 A box cDNA fragments in the use, reclaim the purpose fragment.

(4) behind use KpnI, two kinds of restriction enzymes double zyme cutting pUC19 of the HindIII carrier, reclaim and cut carrier.

(5) use dna ligase to connect above-mentioned (1)-(3) gained fragment, transform in intestinal bacteria, screening positive clone makes up people's HMGB1 A box/ restriction endonuclease sites (EcoR I)/TMPD1 on the pUC19 carrier.

(6) be template with the DNA that clones coding people HMGB1 A box/ restriction endonuclease sites (EcoR the I)/TMPD1 on the pUC19 carrier, through a step inverse PCR strategy, in disappearance restriction endonuclease sites encoding sequence, people HMGB1 A box is directly linked to each other with the encoding sequence of TMPD1.

(7) the people HMGB1 A box of clone on the pUC19 carrier of above acquisition is subcloned on prokaryotic expression carrier pQE 80L with fragment that TMPD1 directly links to each other and transforms in bacillus coli DH 5 alpha, induce through IPTG, expressing protein, through SDS-PAGE and Western blot exist position, the expression level etc. of albumen in the host bacterium are carried out Analysis and Identification, adopt Ni ²⁺-NTA affinity chromatography system carries out purifying to albumen.Be aided with the molecular sieve purification strategy in case of necessity, so that further improve the purity of target protein.

1. the clone (Fig. 1) of people TMPD1/HMGB1 A box gene

1.1 separation of human peripheral blood lymphocytes

(1) gets anticoagulation 5ml, with 1: 1 mixing of PBS damping fluid.

(2) get lymphocyte separation medium (Chinese T BD biotech development center) 10ml.

(3) carefully (1) gained mixed solution is added on (2) gained parting liquid liquid level with dropper, room temperature was placed 5 minutes.

(4) the 2000rpm level is centrifugal 20 minutes, collects the tunica albuginea layer.

(5) PBS damping fluid washed cell twice, centrifugal 10 minutes of 2000rpm abandons supernatant, and the gained precipitation contains the human peripheral blood mononuclear cell.

1.2 the extraction of cell total rna

Adopt Trizol to extract cell total rna, operation steps is undertaken by Trizol reagent specification sheets.

(1) in the gained cell, according to 1 * 10 ⁷Add 1ml Trizol liquid in the individual cell, blow and beat lysing cell repeatedly.

(2) room temperature was placed 5 minutes, made the abundant cracking of cell.

(3) add the 0.5ml chloroform, thermal agitation 15 seconds, room temperature was placed 5 minutes.

(4) 4 ℃, centrifugal 15 minutes of 12000rpm, liquid is divided into three layers.

(5) get colourless water and move in another clean EP pipe, add the equivalent Virahol and put upside down mixing, room temperature was placed 10 minutes.

(6) 4 ℃, centrifugal 10 minutes of 12000rpm abandons supernatant.

(7) add 75% washing with alcohol RNA precipitation.

(8) 4 ℃, centrifugal 5 minutes of 7500rpm abandons supernatant.Add the absolute ethanol washing precipitation, be beneficial to and remove moisture as early as possible.

(9) add 20 μ l DEPC water dissolution precipitation, ultraviolet spectrophotometer is surveyed its concentration.

1.3RT-PCR

1.3.1 reverse transcription synthesizes cDNA

1.3.2?PCR

The segmental condition of amplification TMPD1: pre-sex change in 94 ℃ * 2 minutes; (94 ℃ * 30 seconds, 60 ℃ * 30 seconds, 72 ℃ * 1 minute) * 30 circulations; 72 ℃ * 10 minutes; The segmental condition of amplification HMGB1 A box: pre-sex change in 95 ℃ * 2 minutes; (95 ℃ * 30 seconds, 54 ℃ * 45 seconds, 72 ℃ * 1 minute) * 25 circulations; 72 ℃ * 5 minutes; Primer sequence and amplified fragments see Table 1.Used reaction system is as follows:

Table 1 PCR primer

1.4 make up the order-checking plasmid

1.4.1 make up the people TMPD1/HMGB1 A box fusion gene that links to each other via restriction endonuclease sites

After above-mentioned PCR product is used respective limits restriction endonuclease digestion separately, PCR product that electrophoresis recovery enzyme is cut and the pUC19 carrier of cutting through the KpnI/HindIII enzyme (Fig. 2).Use the T4 ligase enzyme to connect purpose fragment and carrier, transform (classical CaCl in competence DH5 α ₂The method preparation), make up people's TMPD1/ restriction endonuclease sites (EcoR I)/HMGB1 A box on the pUC19 carrier.

1.4.2 make up the people TMPD1/HMGB1 A box fusion gene that directly links to each other

With the DNA that clones coding people TMPD1/ restriction endonuclease sites (EcoR the I)/HMGB1 A box on the pUC19 carrier is template, through a step inverse PCR strategy, in disappearance restriction endonuclease sites sequence, people HMGB1 A box is directly linked to each other with the encoding sequence of TMPD1.

The concise and to the point process following (Fig. 3) of one step inverse PCR method:

(1) designs a pair of primer in opposite direction in the both sides for the treatment of the deletion mutantion position, make the starting point of upstream primer 5 ' end move length to be lacked to 3 of corresponding primer ' end.Upstream primer: 5 '-CCTGCAGGATGGGCAAAGG-3 ' (SEQ ID NO:7); Downstream primer: 5 '-TGGCTGGGAAGTGGAACTC-3 ' (SEQ ID NO:8).

(2) be template with the DNA that clones coding people TMPD1/ restriction endonuclease sites (the EcoRI)/HMGB1 A box on the pUC19 carrier, utilize the high-fidelity DNA polymerase amplification to comprise the recombinant DNA molecules of carrier.Reaction conditions: pre-sex change in 94 ℃ * 5 minutes; (94 ℃ * 30 seconds, 60 ℃ * 30 seconds, 72 ℃ * 5 minutes) * 30 circulations; Reaction system is as follows:

(3) the PCR product is carried out 5 ' terminal phosphateization.

(4) under the effect of dna ligase, finish the recirculation of molecule and transforming in bacillus coli DH 5 alpha.

(5) picking transform bacteria mono-clonal amplification cultivation, extract the recombinant plasmid in each clone source respectively and wherein cloned sequence is carried out determined dna sequence (Fig. 4), therefrom filter out the recon (recombinant plasmid that promptly contains the purpose mutants cDNA) that contains the purpose sudden change.

1.5 construction expression plasmid

The pUC19-TMPD1/HMGB1 A box plasmid that (1) will check order correct after 1.5% agarose gel electrophoresis separates, reclaims purpose fragment through E.Z.N.A.Gel Extract Kit purifying with the KpnI/HindIII double digestion.

(2) with pQE 80L plasmid with KpnI/HindIII double digestion (Fig. 5), the row agarose gel electrophoresis that takes a morsel is observed enzyme and is cut effect, residual enzyme is cut product and is reclaimed with E.Z.N.A.Gel Extract Kit purifying.

(3) enzyme is cut and the good purpose fragment of purifying is connected 12 hours for 16 ℃ with carrier respectively, the connection product transform in the competence bacillus coli DH 5 alpha, cut evaluation through enzyme, prokaryotic expression plasmid pQE80L-TMPD1/HMGB1 A box (Fig. 6) obtains recombinating.

2. the abduction delivering of target protein and evaluation

2.1 the expression of target protein

(1) picking contains single bacterium colony of the bacillus coli DH 5 alpha of reorganization prokaryotic expression plasmid pQE80L-TMPD1/HMGB1 A box and control plasmid pQE 80L, be inoculated in respectively in the 3ml LB liquid nutrient medium (containing 50 μ g/ml Amp), 37 ℃, 225rpm, shaking culture is spent the night.

(2) the bacterium liquid that shaking culture is spent the night is inoculated in respectively in the 5ml LB liquid nutrient medium (containing 50 μ g/ml Amp) by 1%, and 37 ℃, 250r/min shaking culture 3 hours are to mid-log phase [D (600) ≈ 0.6].

(3) adding IPTG respectively is 0.5mmol/L to final concentration, 37 ℃, 250r/min abduction delivering 4 hours.

(4) the proteic expression of SDS-PAGE testing goal.

2.2 denaturing polyacrylamide gel electrophoresis (SDS-PAGE)

(1) respectively get bacterium liquid 1ml behind the abduction delivering, the centrifugal 1min of 10000 * g abandons supernatant.

(2) add each resuspended precipitation of 100 μ l of 1 * SDS-PAGE sample-loading buffer, boiling water bath 10 minutes.

(3) record SDS-PAGE glue with reference to " modern molecular biology experimental technique ".5% spacer gel, 15% separation gel, 1 * SDS Tris-Gly electrophoretic buffer.

(4) respectively get sample on the 20 μ l samples, electrophoresis.Electrophoresis takes off glue, coomassie brilliant blue staining after finishing.Gel imaging instrument record result and the proteic expression of analysis purposes.

2.3 the optimization of abduction delivering condition

Expression condition to pQE80L-TMPD1/HMGB1 A box is optimized

(1) shaking culture the is spent the night bacillus coli DH 5 alpha bacterium liquid that contains reorganization prokaryotic expression plasmid pQE80L-TMPD1/HMGB1 A box is inoculated in respectively in the 5ml LB liquid nutrient medium (containing 50 μ g/mlAmp) by 1%, 37 ℃, 250r/min shaking culture 3 hours are to mid-log phase [D (600) ≈ 0.6].

(2) add respectively IPTG to final concentration be 0.1,0.5,1.0 and 2.0mmol/L.

(3) 37 ℃, 250r/min abduction delivering 4 hours.

(4) SDS-PAGE detects proteic expression, determines the optimum concn that the IPTG inducible protein is expressed.

(5) abduction delivering is carried out according to the best IPTG induced concentration of determining in repeating step (1)-(4), gets each 1ml of culture that added behind the IPTG 1-5 hour respectively, detects proteic expression through SDS-PAGE, determines the Best Times of abduction delivering.

(6) repeating step (1)-(4) according to best IPTG induced concentration and the induction time determined, make and express bacterium respectively 25 ℃, 29 ℃, 33 ℃, 37 ℃ growths, determine the optimum temps of abduction delivering.

2.4 protein immunoblotting (Western Blotting)

(1) the bacillus coli DH 5 alpha bacterium liquid that contains reorganization prokaryotic expression plasmid pQE80L-TMPD1/HMGB1 Abox and control plasmid pQE80L that shaking culture is spent the night is inoculated in respectively in the 5ml LB liquid nutrient medium by 1%, 37 ℃, 250r/min shaking culture 3 hours are to mid-log phase [D (600) ≈ 0.6].

(2) after 4 hours, get 1ml bacterium liquid through 37 ℃ of abduction deliverings of 0.5mmol/L IPTG respectively, centrifugal 1 minute of 10000 * g abandons supernatant, adds each resuspended precipitation of 100 μ l of 1 * SDS-PAGE sample-loading buffer, boiling water bath 10 minutes.Respectively get the capable SDS-PAGE of sample on the 15 μ l, method is the same.

(3) after electrophoresis finishes, take off glue, the excision redundance is put into transfering buffering liquid and is soaked.Cutting 1 simultaneously puts into transfering buffering liquid with the equirotal nitrocellulose filter of gel and 8 filter paper slightly littler than gel and soaks.

(4) transfer device is installed: successively 4 filter paper, nitrocellulose filter, gel, other 4 filter paper are placed on the plastic stent of sponge, all remove bubble when keeping flat for every layer, clamp with plastic stent at last, put into the electrotransfer groove, nitrocellulose filter one side is by anodal, and gel one side is by negative pole.

(5) connect power supply, constant voltage 15V, room temperature shifted 1 hour.

(6) after transfer finishes, remove each layer successively, gel uses coomassie brilliant blue staining to check transfer effect.Nitrocellulose filter dyes with Ponceau S after using the good direction of marking pen mark, and the colour developing back marks the Marker position with marking pen, washes film until complete flush away Ponceau S with distilled water then.

(7) the TBS damping fluid was washed film 10 minutes.

(8) closed 12 hours with the damping fluid room temperature lower seal of blockading, seal non-specific combination site.

(9) wash film twice under the TBS-Tween/Triton damping fluid room temperature, each 10 minutes.

(10) wash film twice under the TBS damping fluid room temperature, each 10 minutes.

(11) add mouse-anti people His antibody (one is anti-) (containing 1000 times of 3%BSA TBS buffer dilutions), horizontal jog is 1 hour under the room temperature, and a target protein and a resistive connection are closed.

(12) wash film twice with the TBS-Tween/Triton damping fluid under the room temperature, each 10 minutes.

(13) wash film twice under the TBS damping fluid room temperature, each 10 minutes.

(14) add the sheep anti-mouse igg (two is anti-) (containing 100 times of 3%BSA TBS damping fluid dilutions) of HRP mark, horizontal jog is 1 hour under the room temperature, makes anti-and two anti-reactions.

(15) wash film 4 times with the TBS-Tween/Triton damping fluid under the room temperature, each 10 minutes, anti-to remove unconjugated two.

(16) by specification adds developer DAB colour developing 15 minutes, observations (Fig. 7).

3. the isolation and purification of target protein

3.1 the great expression of target protein

(1) bacillus coli DH 5 alpha that will contain reorganization prokaryotic expression plasmid pQE80L-TMPD1/HMGB1 A box is inoculated in the 5ml LB nutrient solution (containing 50 μ g/ml Amp) 37 ℃, 200rpm shaken overnight.

(2) the bacterium liquid with shaken overnight is inoculated in the LB nutrient solution that 200ml contains 100 μ g/ml penbritins respectively by 1%, and 37 ℃ of thermal agitations 3 hours are to mid-log phase [D (600) ≈ 0.6].

(3) add IPTG to final concentration 0.5mmol/L.

(4) 37 ℃ are continued vibration 4 hours.

(5) 4 ℃, 4 000g collection in centrifugal 15 minutes bacterium.

3.2 the evaluation of expression of recombinant proteins mode

(1) every gram bacterium adds 3ml TE buffer (pH8.0).

(2) carrying out ultrasonic bacteria breaking becomes limpid until heavy-gravity bacterium liquid in ice bath.

Centrifugal 15 minutes of (3) 4 ℃, 4 000g are collected respectively and are gone up cleer and peaceful precipitation, respectively take a morsel to add the SDS-PAGE sample-loading buffer and carry out the SDS-PAGE electrophoresis, and be to express to determine fusion rotein with secreted form, still form inclusion body (Fig. 7).

3.3 washing inclusion body

(1) supernatant is put 4 ℃ of preservations temporarily.Precipitation is resuspended with the lavation buffer solution of 9 times of volumes, and room temperature was placed 5 minutes.

Centrifugal 15 minutes of (2) 4 ℃, 12 000g.

(3) repeat aforesaid operations once, the PMSF that adds final concentration in the supernatant and be 1mmol/L puts 4 ℃ of preservations temporarily.

3.4 dissolving inclusion body

(1) will precipitate adding 4ml buffer A, and add PMSF to final concentration 0.1mmol/L.

(2) stir 60 minutes, until the solution shape that is translucent.

Centrifugal 30 minutes of (3) 10 000g, supernatant adds PMSF once more, and precipitation is put 4 ℃ of preservations temporarily.

3.5 the purifying of target protein (being undertaken) by Qiagen company specification sheets

Because TMPD1/HMGB1 A box albumen is with the inclusion body formal representation, so we carry out purifying according to the albumen of inclusion body form.

(1) gets 1ml 50%Ni ²⁺-NTA slurry adds 4ml solubilization of inclusion bodies thing supernatant, and adds PMSF to final concentration 0.1mmol/L, vibrate gently mixing 60 minutes of room temperature.

(2) said mixture is adorned post, Ultraviolet Detector detects.

(3) open clip, the control flow velocity is collected effluent liquid and is put 4 ℃ of preservations at 0.2ml/min.

(4) wash post with Buffer A, the back absorbancy occurs less than 0.01, collect effluent liquid and put 4 ℃ of preservations until absorption peak.

(5) wash post with Buffer B, the back absorbancy occurs less than 0.01, collect effluent liquid and put 4 ℃ of preservations until absorption peak.

(6) wash post with Buffer C, the back absorbancy occurs until absorption peak and no longer change (because imidazoles itself has certain light absorption value), collect elutriant and add the rearmounted 4 ℃ of preservations of PMSF.

(7) wash post with Buffer D, less than 0.01, collect elutriant and put 4 ℃ of preservations until absorbancy.

3.6 dialysis renaturation

(1) prepares dialysis tubing: containing 2%NaHCO ₃With boiled 10 minutes in the solution of 1mmol/L EDTA, rinse well with deionized water, in the solution of 1mmol/L EDTA, boil 10min again, be soaked in the mid-4 ℃ of preservations of 1mmol/L EDTA at last.

(2) be diluted to 50ml after with Buffer A Buffer C and Buffer D elutriant being mixed, stirring and evenly mixing adds PMSF to final concentration 0.1mmol/L, the dialysis tubing of packing into.

(3) dialysis tubing is put into the beaker that the 1L renaturation solution is housed, 4 ℃, 12 hours, stirred for several times therebetween.

(4) discard renaturation solution, add new renaturation solution, 4 ℃, 10 hours, stir for several times therebetween.

(5) the dialysis tubing taking-up is moved in the beaker that 1L TE damping fluid is housed, 4 ℃, 10 hours, stir for several times therebetween.

(6) liquid after the taking-up renaturation, in the clean small beaker of packing into, lyophilize postposition-70 ℃ preservation.

(7) with the capable SDS-PAGE electrophoresis of freeze dried fusion rotein (Fig. 8).

The preparation of inclusion body lavation buffer solution: 0.5%Triton X-100,10mmol/L EDTA, pH8.0

Protein purification is with the preparation of damping fluid (albumen that purifying exists with the inclusion body form):

Buffer A:100mmol/L NaH ₂PO ₄, 10mmol/LTris, 8mol/L urea, pH8.0

Buffer B: get 200ml BufferA, with HCl adjust pH to 6.3

Buffer C: get 200ml Buffer A, add the 2.72g imidazoles, adjust pH to 5.9

Buffer D: get 200ml Buffer A, with HCl adjust pH to 4.5

The preparation of renaturation solution: 0.8mol/L urea, 0.1mmol/L Sleep-promoting factor B, 0.9mmol/L reduced glutathion, 10mmol/L Tris, pH8.0

The preparation of TE damping fluid: 10mmol/L Tris, 1mmol/L EDTA, pH8.0

4. the biologic activity of target protein is identified

Adding the dissolving of 0.5ml sterilization apirogen water 4.1 a small amount of albumen lyophilized powder is respectively taken out in the preparation of sample, is reference with the bovine serum albumin standard substance, and ultraviolet spectrophotometer is surveyed protein concentration.

4.2 the activity identification of recombinant protein TMPD1/HMGB1 A box

1) cultured mononuclear macrophage strain RAW264.7 (ATCC) being adjusted cell count is 1 * 10 ⁶Individual/ml, be incubated in the 24 porocyte culture plates, the reorganization HMGB1 (final concentration is 1 μ g/ml) that adds same dose earlier adds the reorganization TMPD1/HMGB1 A box of same dose again, compares every group 3 hole with DHFR, DHFR/HMGB1 A box, TMPD1.(DHFR, DHFR/HMGB1 A box, TMPD1 obtain in earlier stage for the contriver laboratory, because of HMGB1 A box unstable expression, so replace HMGB1 A box with this fusion form of DHFR/HMGB1 A box, are contrast with DHFR.)

2) add a cover, 37 ℃, cultivated 4 hours in the 5%CO2 incubator.

3) centrifugal culture plate, 800rpm, 5 minutes.

4) carefully draw supernatant, detect reorganization TMPD1/HMGB1 A box with ELISA and suppress the level that HMGB1 induces monocyte TNF secretion-α, and make comparisons, the results are shown in Figure 9 with DHFR, DHFR/HMGB1 Abox, TMPD1.TNF-α ELISA detects and presses test kit specification sheets operation (R﹠amp; D Systems, USA).

The result shows:

The extracorporeal anti-inflammatory effect detect to show, TMPD1/HMGB1 A box can effectively suppress to have the release of the recombinant human HMGB1 inductive TNF-α of clear and definite proinflammatory effect, and this restraining effect obviously is better than independent reorganization human HMGB 1 A box and TMPD1.

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.

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＜110〉Military Medical Univ No.3, P.L.A

＜120〉based on recombination fusion protein of people TMPD1 and HMGB1 A box and preparation method thereof

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<170>PatentIn?version?3.3

<210>1

<211>240

<212>PRT

＜213〉based on the aminoacid sequence of the recombination fusion protein of people TMPD1 and HMGB1 A box

<400>1

Glu?Pro?Gln?Pro?Gly?Gly?Ser?Gln?Cys?Val?Glu?His?Asp?Cys?Phe?Ala

1 5 10 15

Leu?Tyr?Pro?Gly?Pro?Ala?Thr?Phe?Leu?Asn?Ala?Ser?Gln?Ile?Cys?Asp

20 25 30

Gly?Leu?Arg?Gly?His?Leu?Met?Thr?Val?Arg?Ser?Ser?Val?Ala?Ala?Asp

35 40 45

Val?Ile?Ser?Leu?Leu?Leu?Asn?Gly?Asp?Gly?Gly?Val?Gly?Arg?Arg?Arg

50 55 60

Leu?Trp?Ile?Gly?Leu?Gln?Leu?Pro?Pro?Gly?Cys?Gly?Asp?Pro?Lys?Arg

65 70 75 80

Leu?Gly?Pro?Leu?Arg?Gly?Phe?Gln?Trp?Val?Thr?Gly?Asp?Asn?Asn?Thr

85 90 95

Ser?Tyr?Ser?Arg?Trp?Ala?Arg?Leu?Asp?Leu?Asn?Gly?Ala?Pro?Leu?Cys

100 105 110

Gly?Pro?Leu?Cys?Val?Ala?Val?Ser?Ala?Ala?Glu?Ala?Thr?Val?Pro?Ser

115 120 125

Glu?Pro?Ile?Trp?Glu?Glu?Gln?Gln?Cys?Glu?Val?Lys?Ala?Asp?Gly?Phe

130 135 140

Leu?Cys?Glu?Phe?His?Phe?Pro?Ala?Thr?Cys?Arg?Met?Gly?Lys?Gly?Asp

145 150 155 160

Pro?Lys?Lys?Pro?Arg?Gly?Lys?Met?Ser?Ser?Tyr?Ala?Phe?Phe?Val?Gln

165 170 175

Thr?Cys?Arg?Glu?Glu?His?Lys?Lys?Lys?His?Pro?Asp?Ala?Ser?Val?Asn

180 185 190

e?Ser?Glu?Phe?Ser?Lys?Lys?Cys?Ser?Glu?Arg?Trp?Lys?Thr?Met?Ser

195 200 205

Ala?Lys?Glu?Lys?Gly?Lys?Phe?Glu?Asp?Met?Ala?Lys?Ala?Asp?Lys?Ala

210 215 220

Arg?Tyr?Glu?Arg?Glu?Met?Lys?Thr?Tyr?Ile?Pro?Pro?Lys?Gly?Glu?Thr

225 230 235 240

<210>2

<211>720

<212>DNA

＜213〉based on the nucleotide sequence of the recombination fusion protein of people TMPD1 and HMGB1 A box

<400>2

gagccgcagc?cgggtggcag?ccagtgcgtc?gagcacgact?gcttcgcgct?ctacccgggc 60

cccgcgacct?tcctcaatgc?cagtcagatc?tgcgacggac?tgcggggcca?cctaatgaca 120

gtgcgctcct?cggtggctgc?cgatgtcatt?tccttgctac?tgaacggcga?cggcggcgtt 180

ggccgccggc?gcctctggat?cggcctgcag?ctgccacccg?gctgcggcga?ccccaagcgc 240

ctcgggcccc?tgcgcggctt?ccagtgggtt?acgggagaca?acaacaccag?ctatagcagg 300

tgggcacggc?tcgacctcaa?tggggctccc?ctctgcggcc?cgttgtgcgt?cgctgtctcc 360

gctgctgagg?ccactgtgcc?cagcgagccg?atctgggagg?agcagcagtg?cgaagtgaag 420

gccgatggct?tcctctgcga?gttccacttc?ccagccacct?gcaggatggg?caaaggagat 480

cctaagaagc?cgagaggcaa?aatgtcatca?tatgcatttt?ttgtgcaaac?ttgtcgggag 540

gagcataaga?agaagcaccc?agatgcttca?gtcaacttct?cagagttttc?taagaagtgc 600

tcagagaggt?ggaagaccat?gtctgctaaa?gagaaaggaa?aatttgaaga?tatggcaaaa 660

gcggacaagg?cccgttatga?aagagaaatg?aaaacctata?tccctcccaa?aggggagaca 720

<210>3

<211>29

<212>DNA

＜213〉upstream primer of amplification people TMPD1 cDNA

<400>3

gttggtaccg?ccaagctgca?gcccacagg 29

<210>4

<211>32

<212>DNA

＜213〉downstream primer of amplification people TMPD1 cDNA

<400>4

cgcggaattc?tcaaggcctg?caggaagctg?tg 32

<210>5

<211>28

<212>DNA

＜213〉upstream primer of amplification people HMGB1 A box cDNA

<400>5

gttgaattca?tgggcaaagg?agatccta 28

<210>6

<211>25

<212>DNA

＜213〉downstream primer of amplification people HMGB1 A box cDNA

<400>6

gcgaagcttt?gtctcccctt?tggga 25

<210>7

<211>19

<212>DNA

＜213〉inverse PCR upstream primer

<400>7

cctgcaggat?gggcaaagg 19

<210>8

<211>19

<212>DNA

＜213〉inverse PCR downstream primer

<400>8

tggctgggaa?gtggaactc 19

Claims (7)

1. the recombination fusion protein based on people TMPD1 and HMGB1 A box is characterized in that having the aminoacid sequence shown in the SEQ ID NO:1.

2. the recombination fusion protein based on people TMPD1 and HMGB1 A box according to claim 1 is characterized in that it has the nucleotide sequence shown in the SEQ ID NO:2.

3. the preparation method of the described recombination fusion protein based on people TMPD1 and HMGB1 A box of claim 1 is characterized in that comprising following steps:

1) extracts total RNA of human peripheral blood mononuclear cell and be cDNA with its reverse transcription;

2) be template with the cDNA in the step 1), use the nucleotides sequence shown in the SEQ ID NO:3-4 to classify the cDNA of primer amplification people TMPD1 as;

3) be template with the cDNA in the step 1), use the nucleotides sequence shown in the SEQ ID NO:5-6 to classify the cDNA of primer amplification people HMGB1 A box as;

4) with step 2) cDNA of the people TMPD1 that obtains and the cDNA of the people HMGB1 A box that step 3) obtains respectively enzyme cut the back and be connected with carrier;

5) the step 4) gained is connected product and transform in intestinal bacteria, screening positive clone obtains comprising the recombinant vectors of people HMGB1 A box/ restriction endonuclease sites/TMPD1;

6) being template with the step 5) recombinant vectors, is that primer carries out inverse PCR with the Nucleotide shown in the SEQ ID NO:7-8, in disappearance restriction endonuclease sites encoding sequence, people HMGB1 A box is directly linked to each other with the encoding sequence of TMPD1;

7) the people HMGB1 A box that step 6) is obtained transforms in intestinal bacteria with the PCR product that TMPD1 directly links to each other, the recombinant protein that the abduction delivering acquisition is fused to TMPD1 by people HMGB1 A box.

4. method according to claim 3 is characterized in that in the step 4) using KpnI and EcoRI enzyme to cut step 2) cDNA of the people TMPD1 that obtains.

5. method according to claim 3 is characterized in that in the step 4) using EcoRI and HindIII enzyme to cut step 2) cDNA of the people HMGB1 A box that obtains.

6. method according to claim 3 is characterized in that the restriction endonuclease sites in the step 5) is EcoR I.

7. the described application of recombination fusion protein in the preparation anti-inflammatory drug of claim 1 based on people TMPD1 and HMGB1 A box.

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