CN101891812B

CN101891812B - Mechano growth factor polypeptide, preparation method and application thereof

Info

Publication number: CN101891812B
Application number: CN201010224435A
Authority: CN
Inventors: 宋莉; 董俊丽; 李卓玉; 祝文思; 刘晓辉
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2010-07-09
Filing date: 2010-07-09
Publication date: 2012-10-17
Anticipated expiration: 2030-07-09
Also published as: CN101891812A

Abstract

The invention provides a mechano growth factor polypeptide, a preparation method and an application thereof. The mechano growth factor polypeptide is the polypeptide of 41 amino acids at C end of a mechano growth factor, and the polypeptide is taken as an active region of the mechano growth factor. The preparation method comprises the following steps: cloning a nucleotide sequence of the polypeptide into an expression vector pRSETc to obtain a recombinant expression plasmid pRSETc-MGF41; transfecting escherichia coli with the plasmid to obtain an engineering strain for expressing the mechano growth factor polypeptide; and finally culturing and purifying the engineering strain to obtain the mechano growth factor polypeptide. The polypeptide has the function of promoting cell proliferation, thus the polypeptide can be used for preparing a medicine for promoting cell proliferation.

Description

Mechano growth factor polypeptide

Technical field

The present invention relates to growth factor, specifically belong to Mechano growth factor polypeptide (MGF ₄₁).

Background technology

(Mechano growth factor MGF) is the regulatory factor that a kind of mechanically stretching of discoveries such as Goldspink causes the muscle local increase to Mechano growth factor.Muscle under the stimulation of power, insulin-like growth factor I (Insulin-like growth factor, IGF-I) gene shear to produce two kinds of isomer through selectivity, and is a kind of similar with liver type IGF-I, called after muscularity IGF-I (IGF-I Ea); Another kind of because of very responsive, so called after Mechano growth factor (MGF) to mechanical stimulation.Discover that MGF is not only a kind of growth factor, but also be a kind of reparative factor.The function of MGF, main relevant with its C end structure territory.MGF is not too common under the normal physiological state, but when Skelettmuskel or cardiac muscle expression increase under the state that mechanical load strengthens, participates in the process of repairing behind the muscle injury.MGF does not exist only in Skelettmuskel, the cardiac muscle, finds that in neural system and sclerocyte it has the effect of the damage of repairing yet.Recent study finds that MGF can obviously improve the quantity of stem cell in malnutritive muscle and amyotrophic lateral sclerosis (ALS) muscle, and plays repair; The importing of MGF can make underfed muscle strength raising 35% and have the neural effect of significant protection.Therefore, MGF is with a wide range of applications as the medicine of amyotrophy and muscle/nerve injury reparation.

In order to obtain MGF, the expert has carried out some research work both at home and abroad, but the result is unsatisfactory, because MGF is unstable, the expression in engineering bacteria is prone to form inclusion body, and difficulty is taken pure article.Therefore seek a kind of alternative MGF, and the product of operation acquisition easily is very necessary.

Summary of the invention

The purpose of this invention is to provide a kind of Mechano growth factor polypeptide.

A kind of Mechano growth factor polypeptide (MGF provided by the invention ₄₁) be 41 amino acid whose polypeptide of Mechano growth factor C end, its aminoacid sequence is SEQ ID NO:1.

Coding Mechano growth factor polypeptide (MGF provided by the invention ₄₁) nucleotide sequence form by 126 Nucleotide, comprise that one is read frame and terminator codon TAA.Its nucleotide sequence SEQ ID NO:2.

A kind of coding MGF that contains ₄₁The carrier of nucleotide sequence, it is with coding MGF ₄₁Nucleotide sequence clone the recombinant plasmid pRSETc-MGF that obtains behind the expression vector into ₄₁

A kind of MGF ₄₁Engineering bacteria is prokaryotic expression system, and it contains foregoing carrier.Be with described pRSETc-MGF ₄₁In recombinant plasmid transformed to a kind of bacterium, for example e. coli bl21 (DE3) is built into engineering strain, for example pRSETc-MGF ₄₁/ BL21.

MGF ₄₁The preparation method, comprise the steps:

A) make up pRSETc-MGF ₄₁Expression vector;

B), make up colibacillus engineering strain pRSETc-MGF according to standard law transformed into escherichia coli BL21 ₄₁/ BL21 is seeded in engineering bacteria in the LB substratum that contains penbritin, and 37 ℃, the 150r/min shaking culture is to OD ₆₀₀Be 0.5-0.7, it is 0.5mmol/L that adding IPTG makes final concentration, abduction delivering 5 hours;

C) collect thalline, smudge cells, centrifugal, supernatant is obtained target protein through cation-exchange chromatography, lyophilize.

Adopt the MGF of CCK-8 method to purifying ₄₁BA study.Experiment showed, MGF ₄₁Has the myocyte of facilitating C ₂C ₁₂The activity of propagation, this polypeptide can be used for preparing the medicine of short cell proliferation.

Advantage compared with prior art of the present invention and effect: MGF of the present invention ₄₁The preparation method of polypeptide is simple, and single step purification can obtain target protein, and this protein stability is better, and has the BA of well short cell proliferation.

Description of drawings

Fig. 1 MGF ₄₁Tricine-SDS-PAGE figure.

Fig. 2 MGF ₄₁Short cell-proliferation activity.

Embodiment

Embodiment 1:

MGF ₄₁The structure of expression vector

The pExSecI-MGF that preserves with the laboratory is a template; Utilize Auele Specific Primer: Forward primer:gat at gga tcc gctagc gct cgc tct gtc cgt gcc cag and Reverse primer:cgc gc ctc gag tta ctt gtg ttc ttc aaa tgt ac carry out pcr amplification; The gained amplified production reclaims test kit through OMEGA glue and reclaims goal gene; Use Nhe I and Xho I double digestion then, enzyme is cut product and is connected through the T4 dna ligase with the carrier pRSETc of the same double digestion of warp.Connect product transformed into escherichia coli DH5 α, the picking positive colony carries out bacterium colony PCR and enzyme and cuts evaluation.Through dna sequencing analysis revealed, MGF ₄₁Be cloned into pRSETc, with this recombinant plasmid called after pRSETc-MGF ₄₁

MGF ₄₁Expression, purifying and determination of activity

With recombinant plasmid pRSETc-MGF ₄₁Transformed into escherichia coli BL21 (DE3) makes up colibacillus engineering strain pRSETc-MGF ₄₁/ BL21 is seeded in engineering bacteria in the LB substratum that contains penbritin, selects different inductor concentration (0.1-1.0mmol/L) respectively for use, and induction time (1-6h) and inducing temperature (37 ℃, 30 ℃, 16 ℃) target protein is expressed.Through optimizing, finally be selected in 37 ℃, the 150r/min shaking culture is to OD ₆₀₀Be 0.5-0.7, it is 0.5mmol/L that adding IPTG makes final concentration, and abduction delivering 5 hours carries out the expression of target protein.The final thalline of collecting, smudge cells carries out the Tricine-SDS-PAGE analysis with full bacterium and supernatant, and the result is illustrated in about 5KD place has band of expression, with the MGF that estimates ₄₁Molecular weight of albumen size identical (like Fig. 1, swimming lane 3 and 4).And the target protein major part is expressed with soluble mode.

The soluble-expression albumen supernatant that contains of above-mentioned acquisition is passed through SP Sepharose FF cation-exchange chromatography post, with the PB buffer solution elution that contains 10mM-1M NaCl, collect target protein after the PB damping fluid balance, lyophilize is MGF ₄₁Product.This product is analyzed through Tricine-SDS-PAGE, is single band (like Fig. 1, swimming lane 5), and this shows and has obtained the higher MGF of purity ₄₁Product.

Adopt the MGF of CCK-8 method to purifying ₄₁BA detect, detailed process is following: at 37 ℃, contain 5%CO with DMEM high glucose medium (containing 10% foetal calf serum) ₂Incubator in cultivate the C2C12 cell, make its adherent growth, the C2C12 cell in vegetative period of taking the logarithm carries out serum starvation 24h.With the C2C12 cell of 0.25% trysinization adherent growth, with the DMEM substratum re-suspended cell that does not contain serum, to process cell suspension, and count again with the blood cell counting plate pair cell, the adjustment number of cells is 5 * 10 ⁴Individual/mL.Every hole adds above-mentioned cell suspension 100 μ L in 96 orifice plates.At 37 ℃, contain 5%CO ₂Incubator in hatch up to cell adherent fully.Not contain the DMEM nutrient solution dilution MGF of calf serum ₄₁Sample adds in 96 orifice plates, and sample concentration is respectively 0,5,10,50,500,10000ng/mL, and each concentration repeats 5 holes, and every hole adds 50 μ LMGF samples, does contrast with the BSA with concentration.37 ℃, 5%CO ₂Cultivate 12h under the condition.Every hole adds people's 10 μ LCCK-8 solution, 37 ℃, 5%CO ₂Continue to hatch 2h under the condition.Measure the absorbance at wavelength 450nm place with ELIASA.Detected result such as Fig. 2 show MGF ₄₁Activity with short cell proliferation can be used for preparing the medicine of short cell proliferation.

Claims

1. a Mechano growth factor polypeptide is characterized in that aminoacid sequence is: SEQ ID NO:1.

2. the Nucleotide of Mechano growth factor polypeptide of coding claim 1 is characterized in that nucleotides sequence classifies as: SEQ ID NO:2.

3. carrier, it contains the described nucleotide sequence of claim 2.

4. an engineering bacteria is characterized in that containing the described carrier of claim 3.

5. the said engineering bacteria of claim 4 is characterized in that engineering bacteria is a prokaryotic expression system.

6. the described Mechano growth factor polypeptide of claim 1 is facilitated the application in the muscle cell multiplication medicine in preparation.