CN105200061A - Human recombinant Irisin protein and preparation method and application thereof - Google Patents

Abstract

The invention discloses a DNA sequence of human Irisin recombinant protein, further discloses the human Irisin recombinant protein, further discloses a recombinant expression vector, a transgenic cell system or a transgenic recombinant bacterium of the DNA sequence of the human Irisin recombinant protein, further discloses a preparation method of the human Irisin recombinant protein, and further discloses application of the human Irisin recombinant protein in preparing drug for metabolic diseases. The preparation method is simple and convenient in step, low in production cost and short in production period, and high-purity target protein can be obtained through the preparation method.

Description

A kind of people recombinates Irisin albumen and its preparation method and application

Technical field

The present invention relates to solubility expression and the purification process of recombination in genetically engineered field, particularly the prokaryotic expression of people Irisin albumen and purification process.

Background technology

Irisin is a kind of new muscle factor reported on " nature " magazine by Spiegelman laboratory at the beginning of 2012, be proteolytic ferment shear that III type fibronectin assembly comprises that albumen 5 (fibronectintype III domain-containingprotein5, FNDC5) formed afterwards can secrete polypeptide fragment.People FNDC5 N-end have a signal sequence, centre is III type fibronectin domain (FND), immediately thereafter be hydrophobic amino acid district and C-end.Irisin is the major portion of fibronectin domain in FNDC5, the N-glycosylated protein hormone be made up of 112 amino-acid residues, its sequence high conservative between kind, and the Irisin amino acid sequence homology of people and mouse is 100%.

In discoverer's Greek mythology, the name of courier goddess Iris is Irisin name, suggests it as Iris, as the envoy of skeletal muscle, transmits the signal of skeletal muscle, and connects the relation of skeletal muscle and peripheral tissues.The most important biological function of Irisin acts on white adipocyte by the mode of internal secretion, paracrine and autocrine, the latter is changed have into the brown fat cell of katabolism fatty character, eliminate white adipose in the mode of heat production, thus play antiobesity action.Research shows, irisin level mainly reflects muscle quality, and it is measured higher than old women in the Male movement person of youth; Separately there are some researches show, the serum I risin level of type ii diabetes patient reduces, and be inverse ratio with the new sickness rate sending out type ii diabetes, shows that Irisin may play keying action in sugar tolerance and type ii diabetes; In addition, about report also points out that Irisin and the pathological phenomenon such as non-alcoholic fatty liver disease, heart rate exhaustion exist certain dependency.

Can find out that Irisin will be a very promising active factor, and become the novel targets of metabolic disease and prevention and treatment of complication thereof, have very large potential value.Therefore, can be mass-produced and being convenient to the method for purifying of exploitation Irisin albumen, has very large value.

Summary of the invention

Goal of the invention: first object of the present invention is to provide a kind of DNA sequence dna of people Irisin recombinant protein.

Another object of the present invention is to provide a kind of people Irisin recombinant protein.

Another object of the present invention is to provide the recombinant expression vector of the DNA sequence dna containing described people Irisin recombinant protein, transgenic cell system or transgenosis recombinant bacterium.

Another object of the present invention is to provide a kind of transgenosis recombinant bacterium.

Another object of the present invention is to provide a kind of preparation method of people Irisin recombinant protein.

Another object of the present invention is to provide a kind of people Irisin recombinant protein in the application preparing metabolic disease medicine.

Technical scheme: in order to solve the problem, technical scheme of the present invention is to provide a kind of DNA sequence dna of people Irisin recombinant protein, and its base sequence is as shown in SEQIDNO:1.

A kind of people Irisin recombinant protein, its aminoacid sequence is as shown in SEQIDNO:2.

The recombinant expression vector of the DNA sequence dna containing described people Irisin recombinant protein, transgenic cell system or transgenosis recombinant bacterium.

Described DNA sequence dna is inserted into the recombinant expression vector of the DNA of the expression people Irisin recombinant protein obtained in coli expression carrier.

Wherein, described coli expression carrier is pET-30a (+).

A kind of transgenosis recombinant bacterium, described recombinant bacterium imports in intestinal bacteria by described recombinant expression vector, and screening obtains transgenosis recombinant bacterium.

Described DNA sequence dna, recombinant expression vector, transgenic cell system or transgenosis recombinant bacterium are producing the application in people Irisin recombinant protein.

A preparation method for people Irisin recombinant protein, comprises the following steps:

1) from people's muscle tissue, extract total serum IgE and do reverse transcription PCR and obtain its cDNA, take cDNA as template, PCR obtains the gene fragment of people Irisin, and its base sequence is as shown in SEQIDNO:1;

2) pET30a-Irisin prokaryotic expression carrier is built;

3) Rosetta-pET-30a-Irisin prokaryotic expression bacterial strain is built;

4) abduction delivering, the Isolation and characterization of people Irisin recombinant protein.

A kind of described people Irisin recombinant protein is in the application preparing metabolic disease medicine.

Beneficial effect: the present invention, relative to prior art, has the following advantages: method steps provided by the present invention is easy, and production cost is low, with short production cycle, by method provided by the present invention, can obtain the specificity target protein that purity is higher.Its main advantage is: the present invention achieves the solubility expression of people Irisin albumen first with prokaryotic expression system, and, by imidazole gradient wash-out and pH gradient elution in the process with ni-sepharose purification albumen, determine the optimum washing engaging condition of target protein, highly purified target protein can be obtained.

Accompanying drawing explanation

Fig. 1: pET-30a-Irisin carrier enzyme cuts inspection agarose electrophoretic analysis figure, and (M is DL5000DNAMaker; 1 is the pET-30a-Irisin carrier through NdeI, XhoI double digestion; 2 is the pET30a-Irisin carrier cut without enzyme.Tentatively can judge that pET-30a-Irisin carrier successfully builds.)

(M is PremixedProteinMarker to Fig. 2: Rosetta-pET-30a-Irisin bacterial strain IPTG gradient SDS-PAGE electroph-oresis analysis chart; In figure, 1 IPTG inducer concentrations used is 0mM; 2 IPTG inducer concentrations used are 0.1mM; 3 IPTG inducer concentrations used are 0.2mM; 4 IPTG inducer concentrations used are 0.4mM; 5 IPTG inducer concentrations used are 0.6mM; 6 IPTG inducer concentrations used are 0.8mM; 7 IPTG inducer concentrations used are 1.0mM; Can find out that 14KDa right position has notable difference, tentatively judge that target protein has expression)

(M is PremixedProteinMarker to Fig. 3: Rosetta-pET-30a-Irisin bacterial strain low temperature induction ni-sepharose purification SDS-PAGE electrophoretic analysis figure; In figure, 1 is induction thalline; 2 is induction cellular lysate supernatant; 3 was post liquid; 4 is washings I; 5 is cleaning solution II; 6 is elutriant; 6 swimming lanes are purifying target protein, its band single and with predict the outcome consistent, this shows that the present invention obtains the target protein of higher degree by purifying)

(be 14KDa location specific band in figure, wherein, 1 is the Rosetta-pET-30a-Irisin thalline without IPTG induction to Fig. 4: people Irisin albumen pronucleus expression Westernblot analysis chart; 2 is the Rosetta-pET-30a-Irisin thalline through IPTG induction; 3 is the Rosetta-pET-30a-Irisin cellular lysate supernatant through IPTG induction; 4 is the people Irisin prokaryotic expression protein through ni-sepharose purification.Result shows, target protein successful expression.)

Fig. 5: BCA determination of protein concentration typical curve.

Embodiment

Embodiment 1: the prokaryotic expression plasmid and the bacterial strain that build people Irisin albumen

1. from the goal gene to Irisin people's muscle tissue

From people's muscle tissue, extract total serum IgE and do reverse transcription PCR and obtain its cDNA.Announce Irisin gene order (NM_153756) on the net according to pertinent literature report and NCBI and design primer, with cDNA obtained above for template, do the gene fragment that PCR obtains people Irisin.Above-mentioned people Irisin gene is inserted in pMD19-T carrier.Called after pMD19-T-Irisin, and order-checking is determined correctly to obtain goal gene.

2. build pET30a-Irisin prokaryotic expression carrier

Follow according to people Irisin gene order design primer, wherein upstream is with NdeI restriction enzyme site, and downstream is with XhoI restriction enzyme site:

Upstream primer: 5 '-TACATATGGACAGTCCCTCAGCCCCA-3 '

Downstream primer: 5 '-CACTCGAGCTCTTTCATGGTTACCTCA-3 '

With pMD19-T-Irisin carrier in step 1 for template, carry out pcr amplification with above-mentioned pair of primers.

PCR system:

Reaction conditions is: 95 DEG C of denaturation 2min; 95 DEG C of sex change 30sec, 62 DEG C of annealing 30sec, 72 DEG C extend 40sec, 30 circulations; 72 DEG C extend 10min; 16 DEG C of end.

PCR primer after NdeI, XhoI double digestion, be connected to pET-30a (+) carrier through NdeI, XhoI double digestion, and transformation of E. coli DH5 α competent cell.The LB flat board that DH5 α after conversion is coated with containing kantlex screens, cultivate 12h for 37 DEG C, picking list colony inoculation contains the LB liquid nutrient medium of kantlex to 3mL, 12h cultivated by 37 DEG C of shaking tables, after doing bacterium liquid PCR qualification, extract the qualification of plasmid NdeI, XhoI double digestion, and carry out checking order to determine correct carrier construction, the carrier called after pET-30a-Irisin checking order correct.

3. build Rosetta-pET-30a-Irisin prokaryotic expression bacterial strain

Owing to containing colibacillary rare codon in the gene order of people Irisin, so choosing " omnipotent " bacterial strain Rosetta (DE3) pLysS is expression strain, this bacterial strain supplements the tRNAs of codon AUA, AGG, AGA, CUA, CCC and GGA by a consistency chlorampenicol resistant plasmid, thus strengthens the expression with the eukaryotic protein of intestinal bacteria rare codon.

The pET-30a-Irisin vector built in step 2 is entered Rosetta (DE3) pLysS competent cell, the LB flat board be coated with containing paraxin and kantlex screens, cultivate 12h for 37 DEG C, picking list colony inoculation contains the LB liquid nutrient medium of paraxin and kantlex to 3mL, 12h cultivated by 37 DEG C of shaking tables, after doing bacterium liquid PCR qualification, extracts plasmid, and carry out double digestion qualification with NdeI, XhoI, the Strain Designation after identifying is Rosetta-pET-30a-Irisin.

Table 1 people Irisin gene order rare codon prognostic chart

Embodiment 2: the abduction delivering of target protein, Isolation and characterization

1. with IPTG abduction delivering target protein

(1) gradient induction: the Rosetta-pET-30a-Irisin bacterial strain line purifying built, picking mono-clonal is seeded to 3mL and contains in the LB liquid nutrient medium of paraxin and kantlex, 37 DEG C of shaking table overnight incubation, after bacterium liquid PCR identifies, being seeded to 3mL by 1% amount contains in the LB liquid nutrient medium of paraxin and kantlex, 37 DEG C of shaking tables cultivate 2 ~ 3h to OD600 to 0.4 ~ 1, add IPTG concentration respectively to 0mM, 0.1mM, 0.2mM, 0.6mM, 0.4mM, 0.8mM, 1.0mM, thalline is collected after 37 DEG C of shaking table inducing culture 5h, through SDS-PAGE electroresis appraisal, can tentatively judge target protein abduction delivering.

(2) low temperature induction: the Rosetta-pET-30a-Irisin bacterial strain line purifying built, picking mono-clonal is seeded to 3mL and contains in the LB liquid nutrient medium of paraxin and kantlex, 37 DEG C of shaking table overnight incubation, after bacterium liquid PCR identifies, being seeded to 50mL by 1% amount contains in the LB liquid nutrient medium of paraxin and kantlex, 37 DEG C of shaking tables cultivate 2 ~ 3h to OD600 to 0.4 ~ 1, put into 18 DEG C of shaking tables and cultivate 0.5h, add IPTG concentration to 0.1mM, 8 DEG C of shaking table inducing culture 18h, collect thalline, (low temperature induction can improve the solubility expression of target protein to carry out protein purification and qualification, induction thalline can continue purifying or be kept at-80 DEG C of refrigerators).

2. use ni-sepharose purification target protein

Get the Rosetta-pET-30a-Irisin bacterium liquid of 100mL low temperature induction, with refrigerated centrifuge 4 DEG C, 5000g collects thalline in centrifugal 10 minutes, add 10mL nickel post containing N,O-Diacetylmuramidase in conjunction with liquid, place 30 minutes on ice, ultrasonication 15 minutes, make thalline thoroughly broken, 4 DEG C, 12000g removes residual cell and cell debris in centrifugal 10 minutes, collects supernatant, use ni-sepharose purification soluble proteins, through SDS-PAGE electroresis appraisal, purifying protein band is very single and in the same size with prediction, shows the target protein obtaining higher degree.

3. with Westernblot method qualification target protein

Get the Rosetta-pET-30a-Irisin thalline without IPTG induction respectively, the Rosetta-pET-30a-Irisin thalline through IPTG induction, the people Irisin prokaryotic expression protein that obtains through Rosetta-pET-30a-Irisin cellular lysate supernatant, the ni-sepharose purification of IPTG induction carry out Westernblot qualification, can determine that the present invention successfully constructs the prokaryotic expression bacterial strain of people Irisin albumen, obtain a kind of prokaryotic soluble expression and purification process of people Irisin albumen.

4. measure the target protein rate of recovery by BCA method

With nickel post elutriant, the BSA standard protein of 25mg/mL is diluted to the standard substance of 0.5mg/mL, by standard substance by 0,1,2,4,8,12,16,20 μ L are added in the standard sample wells of 96 orifice plates, add nickel post elutriant and supply 20 μ L, the target protein of ni-sepharose purification is joined in the testing sample hole of 96 orifice plates, every hole adds 200 μ LBCA working fluids, places 30 minutes, measures absorbancy (A by microplate reader for 37 DEG C _562nm), with standard substance absorbancy drawing standard curve (y=1.02107x-0.12591, R ²=0.99815, wherein y is protein concentration mg/mL, x is absorbancy), calculating sample concentration is 0.225mg/mL, finally show that people Irisin protein recovery of the present invention is about 5-10 milligrams per liter of culture.

The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (9)

1. a DNA sequence dna for people Irisin recombinant protein, its base sequence is as shown in SEQIDNO:1.

2. a people Irisin recombinant protein, is characterized in that, its aminoacid sequence is as shown in SEQIDNO:2.

3. contain the recombinant expression vector of the DNA sequence dna of people Irisin recombinant protein according to claim 1, transgenic cell system or transgenosis recombinant bacterium.

4. recombinant expression vector according to claim 3, is characterized in that, DNA sequence dna according to claim 1 is inserted into the recombinant expression vector of the DNA of the expression people Irisin recombinant protein obtained in coli expression carrier.

5. recombinant expression vector according to claim 4, is characterized in that, described coli expression carrier is pET-30a (+).

6. a transgenosis recombinant bacterium, is characterized in that, described recombinant bacterium imports in intestinal bacteria by the recombinant expression vector described in claim 4 or 5, and screening obtains transgenosis recombinant bacterium.

7. DNA sequence dna according to claim 1, recombinant expression vector according to claim 3, transgenic cell system or transgenosis recombinant bacterium are producing the application in people Irisin recombinant protein.

8. a preparation method for people Irisin recombinant protein, is characterized in that, comprises the following steps:

1) from people's muscle tissue, extract total serum IgE and do reverse transcription PCR and obtain its cDNA, take cDNA as template, PCR obtains the gene fragment of people Irisin, and its base sequence is as shown in SEQIDNO:1;

2) pET30a-Irisin prokaryotic expression carrier is built;

3) Rosetta-pET-30a-Irisin prokaryotic expression bacterial strain is built;

4) abduction delivering, the Isolation and characterization of people Irisin recombinant protein.

9. a people Irisin recombinant protein according to claim 2 is in the application preparing metabolic disease medicine.