CN102206282B - Efficient method for producing Trx-hCTRP2 - Google Patents

Abstract

The invention discloses an efficient method for producing Trx-hCTRP2. The method mainly comprises the following steps of: (1) cloning a human hCTRP2 gene; (2) constructing a prokaryotic expression vector by carrying out a double digestion on an expression vector pET32-a (+) and a plasmid hCTRP2/ pMD20-T with XhoI and BamH I, purifying for recovery, and utilizing a T4DNA ligase for connection to obtain a recombinant vector pET32/ hCTRP2; (3) conversing the recombinant vector into a escherichia coli host; (4) carrying out soluble induction expression; (5) purifying. According to the method of the invention, pET32 is employed as the vector and inducible expression conditions are optimized, so as to highly express a soluble hCTRP2 fusion protein. The method not only guarantees biological activities of the hCTRP2 fusion protein, but also enables efficient obtainment of a large amount of stabilized proteins.

Description

The method of High-efficient Production Trx-hCTRP2 a kind of

Technical field

The present invention relates to use recombinant DNA technology producer gene engineered protein drug technique, relate in particular to a kind of High-efficient Production and also have histidine-tagged thioredoxin (Trx)-hCTRP2 fusion rotein.

Technical background

Adiponectin is a kind of cytokine that is produced by the adipocyte secretion, and nineteen ninety-five is by discoveries first in the 3T3 of mouse adipocyte such as Scherer.Studies show that, it has increase Fatty Acid Oxidation, raising glucose uptake amount, improves the effects such as inflammatory reaction of insulin resistant, modulating vascular endothelium, and closely related with obesity, diabetes B and cardiovascular disorder.CTRPs (Clq-tumor necrosis factor-related protein) is a class and adiponectin structure and function very similar novel protein family, 12 members' (CTRP1-12) that in ncbi database, total this family has found cDNA and protein sequence.CTRPs albumen is comprised of four different structural domains: a N-end signal peptide, a short variable domains, a collagen structure territory, and the C-end globosity territory with complement proteins C1q homology.The discoveries such as Wong, CTRPs is the glycoprotein that can secrete in Mammals.And CTRPs only expresses at fatty tissue unlike adiponectin, and they can be expressed in Various Tissues.

CTRPs has high conservative property.Mouse CTRPs and corresponding people's CTRPs hold the variable region that the amino acid consistence of 53%-100% is arranged at N-, have reached 82%-99% in the such consistence of C-end.In CTRPs (CTRPs) 1-7, the biological function of CTRP2 and structure are all similar to adiponectin.In C2C12 myotube cell, CTRP2 also can quick active AMPK, ACC, the p44/42MAPK approach.As the adiponectin stimulation C2C12 myotube cell 16h with full-length CTRP2, CTRP2 globular domain (gCTRP2) or prokaryotic expression, the accumulation volume that can observe liver starch has risen 6-8 doubly.CTRP2 also can significantly promote the oxidation of lipid acid.Wong etc. think, CTRP2 can be used as a kind of potential euglycemic agent.CTRP2 has very high conservative property in the species such as people, mouse, rat, ox, pig, zebra fish.

Along with to the deepening continuously of CTRP mechanism of action, people CTRP2 is as medicine, and its demand also increases greatly.At present, nobody's hCTRP2 albumen of recombinating also on market, animal or insect cell only can be expressed hCTRP2 low-levelly.This study group utilizes yeast expression system that it is expressed, although hCTRP2 can be in yeast secreting, expressing, but the hCTRP2 that expresses is very unstable in yeast, in case namely degrade in secretion table nutrient solution, therefore can't obtain the hCTRP2 albumen (referring to Fig. 8) of total length and homogeneous.Colibacillary expression product exists with the inclusion body form usually, to get complex operations by the sex change renaturation and just can obtain the hCTRP2 of activity form, thereby reduced the productive rate of activated protein, the present application people also utilizes a lot of colibacillary other expression vectors such as pET28-a (+), it has been carried out abduction delivering, find that the product of expressing is inclusion body protein, does not have the hCTRP2 (referring to Fig. 9) of soluble form; At last, obtain highly purified albumen often needs to process through the multistep purification process, and the step of purifying is more, and the yield of protein will be lower, and more may cause the inactivation of target product.Therefore, existing market also nobody's hCTRP2 albumen of recombinating sell.

Summary of the invention

A kind of method that the purpose of this invention is to provide High-efficient Production Trx-hCTRP2 can obtain a large amount of stable, solvable and have a bioactive Trx-hCTPR2 fusion rotein according to the method.

The technical scheme that solves above-mentioned purpose is as follows:

The method of High-efficient Production Trx-hCTRP2 a kind of mainly comprises the following steps:

(1) human cloning hCTRP2 gene: take total mRNA of extracting from people's adipocyte as template, first with the total cDNA of RT-PCR amplification, again take this cDNA as template, amplify complete hCTRP2 gene fragment with special primer, introduce BamH I restriction enzyme site in hCTRP2 special primer used and introduce Xho I restriction enzyme site in end, reclaim the PCR product and be connected to plasmid pMD20-T carrier, get plasmid hCTRP2/pMD20-T, be defined as the correction sequence through order-checking;

(2) build prokaryotic expression carrier: with expression vector pET32-a (+) and plasmid hCTRP2/pMD20-T process Xho I and BamH I double digestion and purifying recovery, and utilize the T4DNA ligase enzyme to connect, get recombinant vectors pET32/hCTRP2;

(3) transform recombinant vectors in escherichia coli host: utilize the T4DNA ligase enzyme to connect, get recombinant vectors pET32/hCTRP2 and be transformed in intestinal bacteria TOP10 bacterial strain, then extract recombinant vectors pET32/hCTRP2 from TOP10; Use the heat shock method, recombinant vectors pET32/hCTRP2 is transferred in the escherichia coli expression bacterial strain (is preferably BL21 (DE3) bacterial strain), obtain including BL21 (DE3) transformant of recombinant vectors pET32/hCTRP2 through the screening of LB Amp resistant panel;

(4) solvable abduction delivering: intestinal bacteria recombinant conversion that obtains is cultured to OD at 37 ℃ ₆₀₀Adding concentration during for 0.45-0.65 is the IPTG of 0.2mM-0.5mM, induces more than 4 hours the abduction delivering recombinant protein body for 37 ℃;

(5) utilize the method for nickel affinity chromatography and molecular sieve to carry out purifying to hCTRP2 albumen, obtained highly purified hCTRP2 soluble protein.

Preferably, the method of described nickel affinity chromatography and molecular sieve is carried out purifying to hCTRP2 albumen: the thalline of collecting the expression bacterium after IPTG induces, with the resuspended liquid of thalline in buffer A (50mM Tris-HCl, 300mM NaCl, 20mMimidazole and 3mM protease inhibitor PMSF, pH 8.0) carry out fragmentation with Ultrasonic Cell Disruptor; Bacterium liquid after fragmentation carries out centrifugal; Centrifugal resulting supernatant liquor joins in the nickel affinity chromatography column of buffer A pre-equilibration; Through buffer B (50mM Tris-HCl, 300mM NaCl, 50mM imidazole and 3mM PMSF, pH 8.0) after rinsing protein purification pillar, add successively different 150mM, the imidazoles buffer C of 200mM and 250mM concentration (50mM Tris-HCl, pH 8.0,300mMNaCl, 3mM PMSF) albumen is eluted, after the albumen of wash-out takes off intracellular toxin, utilize SuperdexG-75 to carry out further purifying to it, obtain highly purified recombinant protein.

Preferably, described hCTRP2 special primer is:

hCTRP2-for：5’-GCCTCGAGAAAAGAGACCCACTGCTTGGCGC-3’

hCTRP2-rev：5’-GCTCTAGATA?CCTCGTTGGGGTCATC-3’。

Trx-hCTRP2 production method of the present invention, screening through inventor's great many of experiments, finally utilize pET32 as carrier, optimum combination abduction delivering condition, method that finally can the solvable hCTRP2 fusion rotein of high efficient expression had both guaranteed that the biologic activity of hCTRP2 fusion rotein had also obtained a large amount of stabilize proteins efficiently.

Method with production hCTRP2 of the present invention has significant advantage: the one, and expression product and solubilising factor Trx merge, and can obtain the hCTRP2 albumen of soluble form; The 2nd, in this expression system, the disulfide linkage that hCTRP2 albumen is folding by suitable mode, formation is correct also keeps native conformation; The 3rd, expression product is found out the method for the active restructuring of an effective purifying hCTRP2 with the HIS label; The 4th, measured the biological activity of the Trx-hCTRP2 of escherichia coli expression, measurement result shows: animal experiment and test cell line prove that all the Trx-hCTRP2 of expression has good biological activity.

Description of drawings

Fig. 1 is hCTRP2 gene PCR result schematic diagram of the present invention;

Fig. 2 is vector construction schematic diagram of the present invention;

Fig. 3 is that the enzyme of expression vector of the present invention is cut and tested spot figure;

Fig. 4 is the optimization figure of expression product of the present invention;

Fig. 5 is purifying and the proof diagram of expression product of the present invention;

Fig. 6 is the cell levels activation analysis figure of expression product of the present invention;

Fig. 7 is the horizontal activation analysis figure of the animal of expression product of the present invention;

Fig. 8 is that the inventor utilizes the WB of the product that Yeast expression carrier obtains to analyze picture;

Fig. 9 is the result schematic diagram that the inventor obtains as expression vector with pET28-a (+).

Embodiment

The present invention selects escherichia coli expression bacterial strain BL21 (DE3), carrier amplification bacterial strain TOP10 and expression vector pET32-a (+) all available from American I nvritrogen company.

The used medium formula is as follows:

1) LB liquid nutrient medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, autoclaving, room temperature preservation;

2) LB/Amp is dull and stereotyped: NaCl 10g, and peptone 10g, yeast extract 5g, distilled water 1L, agar powder 15g after autoclaving, is cooled to below 70 ℃, adds 1mLAmpicillin (100mg/ml), the rear a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices that fully is mixed, 4 ℃ keep in Dark Place;

3) LB/Amp substratum: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L after autoclaving, is cooled to below 70 ℃, adds 1mLAmpicillin (100mg/ml), fully be mixed, 4 ℃ of preservations.LB liquid nutrient medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, autoclaving, room temperature preservation;

4) 50 * TAE agarose gel electrophoresis damping fluid: Tris alkali 121g, glacial acetic acid 28.6mL, 0.5mol/L EDTA (pH8.0) 50mL, adding distil water is settled to 500mL, room temperature preservation;

5) the 50mg/mL penbritin is preserved liquid: penbritin 0.5g, adding distil water dissolve and are settled to 10mL, after packing in-20 ℃ of preservations;

6) 5 * SDS-PAGE sample-loading buffer: 1M Tris-HCl (pH 6.8) 1.25mL, SDS 0.5g, BPB 25mg, glycerine 2.5mL, be settled to 5mL after adding deionized water dissolving, in room temperature preservation, use every part to add 25 μ L beta-mercaptoethanol mixings after packing (approximately 500 μ L is every part);

7) 5 * SDS-PAGE electrophoretic buffer: Tris 15.1g, glycine 94g, SDS 5.0g adds approximately 800mL deionized water, fully is settled to 1L, room temperature preservation after stirring and dissolving;

8) coomassie brilliant blue R_250 staining fluid: coomassie brilliant blue R_250 0.25g, add glacial acetic acid, the 225mL deionized water of 225mL methyl alcohol, 46mL and stir, after filter paper is removed particulate matter, room temperature preservation;

9) Xylene Brilliant Cyanine G destainer: glacial acetic acid 50mL, methyl alcohol 150mL, deionized water 300mL, after fully mixing, room temperature preservation;

The method of the described High-efficient Production hCTRP2 of the present embodiment mainly comprises the following steps:

1, human cloning hCTRP2 gene: take total mRNA of extracting from people's adipocyte as template, the design pair of primers, take total mRNA of extracting from the human blood leukocyte as template, first with the total cDNA of RT-PCR amplification, amplify whole person's hCTRP2 gene fragment of recombinating with the hCTRP2 special primer, the RT-PCR condition is: 95 ℃ of denaturations, 4min, a thermal cycling; 94 ℃ of thermally denature 30s, 55 ℃ take off fiery 30s, and 72 ℃ are extended 45s, 34 thermal cyclings; 72 ℃ of renaturation 10min.The amplification segment that obtains is connected to pMD20-T carrier (available from Guangzhou TAKARA company), gets plasmid hCTRP2/pMD20-T, uses PCR, and enzyme is cut with nucleic acid sequencing and identified, measures sequence consistent with the sequence of the hCTRP2 of Genebank announcement.

The pcr amplification primer is:

hCTRP2-for：5’-GCCTCGAGAAAAGAGACCCACTGCTTGGCGC-3’(SEQ?ID?NO.1)

hCTRP2-rev：5’-GCTCTAGATA?CCTCGTTGGGGTCATC-3’。(SEQ?ID?NO.2)

The PCR result as shown in Figure 1.

2, build the pET32/hCTRP2 plasmid

1) with Xho I and BamH I double digestion recombinant plasmid hCTRP2/pMD20-T, obtain purpose segment hCTRP2, reaction system following (restriction endonuclease used and damping fluid are all available from Dalian TAKARA company):

2) with Xho I and BamH I double digestion pET32-a (+), obtain the carrier segment, reaction system following (restriction endonuclease used and damping fluid are all available from Dalian TAKARA company):

3) by 1) and 2) rear gained purpose segment of step and carrier segment, DNA gel is regained test kit and is reclaimed, and this test kit is available from Dalian TAKARA company, and concrete operations are undertaken by the test kit specification sheets.Build schematic diagram and see Fig. 2.

4) reclaim the purpose segment and the carrier that obtain and carry out ligation with T4DNA ligase enzyme (available from Dalian TAKARA company), reaction system is as follows:

Get recombinant vectors pET32/hCTRP2.

3, transform recombinant plasmid to intestinal bacteria TOP10

PET32a (+)/hCTRP2 connects product Transformed E .coli TOP10 competent cell, and coating LB Amp is dull and stereotyped, cultivates 12 hours in 37 ℃ of constant incubators; The transformant that grows on the picking culture plate, access 5mLAmp ⁺In/LB liquid nutrient medium, 37 ℃ of shaking table overnight incubation are extracted recombinant plasmid; Recombinant plasmid from different mono-clonal bacterium colonies is carried out enzyme cut, 15 μ L enzymes are cut system:

Enzyme tangent condition: 37 ℃ of water-baths, 3h.

Enzyme is cut product and is detected through agarose gel electrophoresis, identifies clone's result (result as shown in Figure 3).

4, transform recombinant plasmid to escherichia coli expression bacterium BL21 (DE3)

The recombinant vectors pET32/hCTRP2 heat shock of positive colony is transformed into e. coli bl21 (DE3) competent cell, coated plate, cultivated 12 hours in 37 ℃ of constant incubators, i.e. LB Amp resistant panel screening obtains including BL21 (DE3) transformant of recombinant vectors pET32/hCTRP2;

5, solvable abduction delivering: intestinal bacteria recombinant conversion that obtains is cultured to OD at 37 ℃ ₆₀₀Adding concentration during for 0.45-0.65 is the IPTG of 02mM-0.5mM, induces more than 4 hours for 37 ℃ and (according to cost, can be 4-10 hour), the abduction delivering recombinant protein body; (as shown in Fig. 4-B).

The optimization of soluble-expression inductive condition:

Intestinal bacteria recombinant conversion that obtains is cultured to OD at 37 ℃ ₆₀₀The IPTG that adds respectively 0mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM and 1.0mM during=0.5 left and right (0.45-0.65), induced 5 hours for 37 ℃, after ultrasonication, the centrifuging and taking supernatant, add the SDS-PAGE sample buffer, its soluble protein is analyzed, and the best induced concentration of determining IPTG is 0.2mM-0.5mM (shown in Fig. 4-A); Enterobacteria recombinant conversion is cultured to OD at 37 ℃ under best IPTG concentration ₆₀₀Begin abduction delivering during=0.5 left and right, collect thalline in 0h, 1h, 2h, 3h, 4h and 5h, after ultrasonication, the centrifuging and taking supernatant, add the SDS-PAGE sample buffer, its soluble protein is analyzed, determine that recombinant protein body best harvesting time is for inducing about 5 hours;

6, the purifying of expression product

Through (37 ℃ of enlarged culturing and abduction deliverings, 0.2mM IPTG induced 5 hours), the thalline of the expression bacterium of collection after IPTG induces, with the resuspended liquid of thalline in 50ml buffer A (50mM Tris-HCl, 300mM NaCl, 20mMimidazole and 3mM protease inhibitor PMSF, pH 8.0) carry out fragmentation with Ultrasonic Cell Disruptor, power 300W, broken 5s, gap 9s, 90 times; Bacterium liquid after fragmentation carries out centrifugal, and 4 ℃, 30000g, 15min; Centrifugal resulting supernatant liquor joins in the nickel affinity chromatography column of buffer A pre-equilibration; Through 100ml buffer B (50mM Tris-HCl, 300mMNaCl, 50mM imidazole and 3mM PMSF, pH 8.0) after rinsing protein purification pillar, (the 150mM that adds the different concns imidazoles, 200mM and 250mM) buffer C (50mM Tris-HCl, pH 8.0,300mM NaCl, 3mM PMSF) albumen is eluted, utilize the imidazoles of 300mM can obtain purity and reach 90% recombinant protein, the molecular weight of fusion rotein is about 55kDa (shown in Fig. 5-A); Gained albumen is analyzed through WB, and the WB result shows, this albumen is the hCTRP2 fusion rotein, and the molecular weight of fusion rotein is about 55kDa (shown in Fig. 5-B); After the albumen of wash-out takes off intracellular toxin, utilize Superdex G-75 to carry out further purifying (balance liquid is PBS) to it, obtain highly purified recombinant protein (Fig. 5-C); And the transformant of empty carrier pET32-a (+) through induce with above-mentioned identical purifying after, only can obtain Trx albumen, size is about 25kDa (as shown in Fig. 5-D); Through above-mentioned purification step, but the bacterium liquid purifying of 1L abduction delivering obtains the approximately recombinant protein of 15 milligrams (purification efficiency is as shown in table 1)

Table 1Trx-hCTRP2 and purification efficiency table

7, the analysis of biological activity of hCTRP2

A) with the C2C12 cell in the 6cm Tissue Culture Plate, be cultured in containing the DMEM substratum of 10%FBS after this cell is close to be reached more than 90%,, add the DEME substratum that contains 2%DHS to induce differentiation, change liquid every day once, at the 6th day of differentiation, the substratum that does not contain blood through 2 hours DMEM was hungry.Adding final concentration in the above-mentioned 6cm noble cells is the Trx-hCTRP2 fusion rotein of 1 μ g/mL, in cell culture incubator 0,5,10,15, take out a ware cell during 20min, after the PBS washing, add the RIPA lysing cell of 200 μ L, centrifugal collection albumen supernatant; Each time is done respectively 3 repetitions; The albumen supernatant of collecting after concentration, is adjusted each sample protein to same concentrations after measured, after 10%SDS-PAGE, and transferring film, sealing, primary antibodie, two anti-and develop; The WB result shows, under this concentration after 15 minutes the phosphorylation level of ACC and AMPK α obviously improve, and only add the control group of Trx not change (result is as shown in Figure 6); Active cells is measured and is shown, the method is expressed and (thioredoxin) Trx-hCTRP2 of purifying can activate the coherent signal path, and the product of expression and purification has biological activity.

B) experimentation on animals: (C57BL/6) mouse in age in 8-10 week, after 4 hours hunger, body weight injection Trx-hCTRP2 recombinant protein and contrast (PBS) damping fluid by 2 μ g/g, before injection and after injection 1, 2, measured the blood sugar concentration of each mouse in 3 and 5 hours, result as shown in Figure 7, show that through experimentation on animals Trx-hCTRP2 has good hypoglycemic activity, after injection 2 hours, the mouse of injection Trx-hCTRP2 reduces significantly than the control group blood sugar concentration utmost point, this hypoglycemic phenomenon still continues after 5 hours in injection, further confirmed the biological activity of expression product.

Claims (3)

1. the method relevant white ?2(hCTRP2 of egg of a Restruction people C1Q tumour necrosis factor) is characterized in that: mainly comprise the following steps:

(1) clone's hCTRP2 gene: take total mRNA of extracting from people's adipocyte as template, first with the total cDNA of RT-PCR amplification, again take this cDNA as template, amplify complete hCTRP2 gene fragment with special primer, hCTRP2 special primer middle and upper reaches used introduce BamH I restriction enzyme site and Xho I restriction enzyme site is introduced in the downstream, reclaim the PCR product and be connected to plasmid pMD20-T carrier, get plasmid hCTRP2/pMD20-T, be defined as the correction sequence through order-checking;

(2) build prokaryotic expression carrier: with expression vector pET32-a(+) and plasmid hCTRP2/pMD20-T process Xho I and BamH I double digestion and purifying recovery, and utilize the T4DNA ligase enzyme to connect, get recombinant vectors pET32/hCTRP2;

(3) transform recombinant vectors in escherichia coli host: recombinant vectors pET32/hCTRP2 is transformed in intestinal bacteria TOP10 bacterial strain, then extracts recombinant vectors pET32/hCTRP2 from TOP10; With the heat shock method, recombinant vectors pET32/hCTRP2 is transferred to escherichia coli expression bacterial strain BL21(DE3) in, the screening of LB Amp resistant panel obtains including the escherichia coli expression bacterial strain transformant of recombinant plasmid pET32/hCTRP2;

(4) solvable abduction delivering: intestinal bacteria recombinant conversion that obtains is cultured to OD at 37 ℃ ₆₀₀Adding concentration during for 0.45-0.65 is the IPTG of 0.2mM-0.5mM, induces 4-10h for 37 ℃, the abduction delivering recombinant protein body;

(5) utilize the method for nickel affinity chromatography and molecular sieve to carry out purifying to hCTRP2 albumen, obtained highly purified hCTRP2 soluble protein.

2. the method for High-efficient Production recombinant human C1Q tumour necrosis factor associated protein according to claim 1-2(hCTRP2), it is characterized in that, the method of described nickel affinity chromatography and molecular sieve is carried out purifying to hCTRP2 albumen: collect the thalline of the expression bacterium after IPTG induces, thalline is resuspended in buffer A carries out fragmentation with Ultrasonic Cell Disruptor, bacterium liquid after fragmentation carries out centrifugal, centrifugal resulting supernatant liquor joins in the nickel affinity chromatography column of buffer A pre-equilibration, after buffer B rinsing protein purification pillar, add successively different 150mM, the imidazoles buffer C of 200mM and 250mM concentration elutes albumen, after the albumen of wash-out takes off intracellular toxin, utilize Superdex G-75 to carry out further purifying to it, obtain highly purified recombinant protein, described buffer A is pH8.0, 50mM Tris-HCl, 300mM NaCl, 20mM imidazoles and 3mM proteinase inhibitor PMSF solution, buffer B is pH8.0, 50mM Tris-HCl, 300mM NaCl, 50mM imidazoles and 3mM PMSF solution, buffer C is pH8.0, 50mM Tris-HCl, 300mM NaCl, 3mM PMSF solution.

3. the method for High-efficient Production recombinant human C1Q tumour necrosis factor associated protein according to claim 1-2(hCTRP2), is characterized in that, described in step (1), special primer is:

5’-GCCTCGAGAAAAGAGACCCACTGCTTGGCGC-3’；

5’-GCTCTAGATA?CCTCGTTGGGGTCATC-3’。

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