CN102911265A

CN102911265A - Recombination variant of human nerve growth factors and preparation method thereof

Info

Publication number: CN102911265A
Application number: CN2011102235366A
Authority: CN
Inventors: 王革
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-08-05
Filing date: 2011-08-05
Publication date: 2013-02-06
Anticipated expiration: 2031-08-05
Also published as: CN102911265B

Abstract

The invention discloses a recombination variant (rhNGF) of human nerve growth factors and a preparation method thereof. According to the recombination variant of the human nerve growth factors designed by a molecular structure research, ten SSSHPIFHRG amino acids are missed and two GA amino acids are excess at the N end of a peptide fragment, after that a sequence starting from EFSV is same as the normal sequence of the human nerve growth factors. CDNA of rhNGF is obtained with a gene synthesis method, engineering bacteria are built to express, an rhNGF fusion protein is obtained, the rhNGF fusion protein is cut by hydroxylamine and then is purified to obtain the purified rhNGF, and the purified rhNGF has natural biological activity.

Description

Restructuring varient of a kind of growth factor of human nerve and preparation method thereof

Growth factor of human nerve (hNGF) is a kind of biologically active factors that nerve reparation function is had regulating effect in the human body, and to promoting neural growth, the regeneration of injured nerve has decisive role.At present hNGF does not all go public both at home and abroad as medicine, only has China to ratify the NGF clinical application of mouse source, but the threat that mouse source NGF pollutes because of species variation and virus reorganized products substitution at last.Unique approach with genetically engineered Restruction growth factor of human nerve.RhNGF has important clinical value, will fill up the blank of international neural repair medicine.There is nerve injury patients up to a million every year in China, and market outlook are considerable.

We have invented a kind of hNGF restructuring varient that obtains by the protein engineering transformation and preparation method thereof.The growth factor of human nerve end of natural radioactivity is Ser-Ser-Ser-Arg-Pro-Ile-Phe-His-Arg-Gly-Glu-Phe-Ser-Val-Cys-Asn-Ser etc.We are by the further investigation to its molecular structure, the new texture of hNGF restructuring varient has been proposed, this restructuring varient (rhNGF) is characterised in that recombinant expressed hNGF peptide section holds first amino acid to begin to have lacked the S Serine from N, the S Serine, the S Serine, the H Histidine, the P proline(Pro), the I Isoleucine, the F phenylalanine, the H Histidine, the R arginine, 10 amino acid such as G glycine, many G glycine, two amino acid of A L-Ala, and from E L-glutamic acid, the F phenylalanine, the S Serine, the V α-amino-isovaleric acid and backward amino acid begin to meet the hNGF normal sequence, lacked 8 amino acid from the bulk molecule amount.We by the above-mentioned protein engineering transformation that hNGF is carried out to obtain drug effect better, more stable hNGF recombinant mutant (rhNGF), it is the biomolecules with new texture, is beneficial to new drug development and clinical application.

The example explanation:

1. the acquisition of goal gene

In order to obtain the cDNA of people NGF restructuring varient, we adopt the synthetic method of full gene to obtain the cDNA of rhNGF.

The synthetic method of salvage that adopts of full gene is carried out.Gene order design is then according to our molecular designing and the gene order of the hNGF of reference report.5 ' end of the hNGF gene order that we design contains the sequence of Kpn1 restriction enzyme site and coding azanol cleavage site, and 3 ' end contains Xba1 restriction enzyme site and termination codon TGA.The synthetic gene implementation sequence is seen Fig. 1.

Fig. 1 .rhNGF synthetic gene implementation sequence

2. gene sequencing

With the cDNA of the rhNGF of synthetic, insertion vector pThioHis A carries out forward order-checking (sequenator: ABI PRISM) with synthetic sequencing primer.

The collection of illustrative plates that will check order uses tricks to calculate machine-readable order, obtains the cDNA sequence and translates into corresponding amino acid, as a result cDNA and the consensus amino acid sequence of hNGF of our design.Show that the cDNA that we clone the rhNGF that obtains is correct.

3. the structure of expression plasmid

The cDNA of rhNGF is inserted directly expression among the pBV220, and expression product forms insoluble occlusion body, needs obtain activated rhNGF through denature and renature.Because rhNGF contains three pairs of disulfide linkage, renaturation is very difficult, causes productive rate very low.In order to obtain the solution expression with high efficiency of rhNGF, we carry out amalgamation and expression to rhNGF and escherichia coli thioredoxin thioredoxin, and thioredoxin can guide albumen thereafter correctly folding, is solubility expression, the tool natural biological is learned active, has avoided the difficult problem of sex change renaturation.

What cutting method was used is the oxammonium hydrochloride patterning method.Oxammonium hydrochloride is the Asn-Gly peptide bond in the scinderin specifically, produces the N end and is the polypeptide of Gly.We find not contain in the rhNGF polypeptide azanol cleavage site Asn-Gly, still selected azanol cleavage site Asn-Gly be tie point and thioredoxin amalgamation and expression rhNGF.The fusion rotein of expressing discharges rhNGF after cutting with azanol, and its initial amino acid is Gly, and aminoacid sequence after this is consistent with the rhNGF sequence.Gly and Met similar performance be placed on the activity that the N end does not all affect albumen, moreover the N terminal amino acid of hNGF are inessential to activity, and 8 amino acid of its N end at first will be hydrolyzed with practical function in human body.

Azanol is the eubolism product in the human body, and the oxammonium hydrochloride cost is low, and cleavage specificity is good, and cutting efficiency is high, is fit to suitability for industrialized production.

We select plasmid pThioHis A as expression vector (Invitrogen company product), and it contains promotor Ptrc promotor, thioredoxin leading peptide and terminator aspA terminator, and the Amp resistant gene.We insert the fragment of synthetic rhNGF cDNA after with the Kpn1-Xba1 double digestion between the Kpn1-Xba1 of above-mentioned plasmid, are built into expression plasmid pTHNGF.

Host Strains is selected e. coli jm109 (available from Invitrogen company).The genotype of e. coli jm109 is: recA1supE44 endA1 hsdR17 gyrA96 relA1 thi Δ (lac-proAB) F ' [traD36proAB ⁺LacI ^qLacZ Δ M15].

Plasmid pTHNGF is transformed into e. coli jm109, namely consists of engineering bacteria pTHNGF/JM109.It is stored in 15% glycerine, is frozen in-70 ℃, be the original species word bank.

4.rhNGF the calibrating of engineering bacteria

4.1 ne ar, cultural characteristic, physio-biochemical characteristics

From the original species word bank, get the pTHNGF/JM109 engineering bacteria that a glycerine is preserved, be seeded in the LBA substratum, 37 ℃ are swayed cultivation 16 hours, be inoculated into (LB+0.1mg/ml Amp) in the LBA substratum with 10% ratio again, continue to cultivate 3 hours, carry out the following inspection as seed liquor:

4.1.1 ne ar

Behind seed liquor coating slide, gramstaining and microscopy.Engineering bacteria is Gram-negative, and thalline is direct rod shape, edge clear, and the blunt circle in two ends, size is basically identical, has no other living contaminants.

4.1.2 cultural characteristic

Dull and stereotyped with seed liquor line LBA, cultivated 20 hours for 37 ℃, naked eyes as seen: bacterium colony be circle, the smooth of the edge, bacterium colony is full, surface wettability, smooth is creamy white, matrix has no pigment formation.

4.1.3 physio-biochemical characteristics

Can utilize glucose, lactose, glycerine as carbon source, can not utilize fructose, inositol.Indole reaction is positive.V.P. reaction is negative, not gelatin hydrolysate.

4.2 antibiotics resistance feature

Unconverted Host Strains BL21 grows at the LB substratum, and (Amp:0.1mg/ml) do not grow on the LBA substratum.

Engineering bacteria pTHNGF/JM109 after conversion well-grown all on LB substratum and LBA substratum.

Show that engineering bacteria pTHNGF/JM109 has the resistance of penbritin.

4.3 plasmid enzyme restriction atlas analysis

From above-mentioned seed liquor, extract plasmid pTHNGF, carry out enzyme and cut evaluation.All can cut out the small band of 370bp with KpnI-XbaI and KpnI-PstI, can cut out the approximately small band of 340bp with EcoRI-PstI, enzyme is cut qualification result and is all met the plasmid design, illustrates that plasmid pTHNGF makes up correct.

4.4 the expression of engineering bacteria and the acquisition of expression product

The pTHNGF/JM109 seed liquor is inoculated in the LBA substratum with 1% ratio, and 37 ℃ grow to OD ₆₀₀Be about 0.5, add IPTG to 1mM as inductor, induced 5 hours for 37 ℃, the results thalline, the SDS-PAGE electrophoretic analysis is carried out in the sampling cracking after the washing.As seen the thalline after inducing has more a dense protein band at the 26kD place than blank, and with the rhNGF fusion protein molecule amount 26kD equal and opposite in direction that will express, through thin layer scanning, this band accounts for about the 10-20% of bacterial protein.

The thalline of expressing is carried out carrying out ultrasonic bacteria breaking, and high speed centrifugation.To precipitate with supernatant and carry out the SDS-PAGE electrophoresis.The rhNGF fusion rotein of as seen expressing concentrates in the supernatant, namely expresses with soluble form, and the tool natural radioactivity does not need through denature and renature.

Method through osmotic pressure discharges is discharged into the solvable rhNGF fusion rotein major part in the Bacillus coli cells in the damping fluid, after the azanol cutting, through Ni chelating chromatography column and CM-Sepharose chromatography column purifying, namely obtains the rhNGF of purifying.

5. expression product structural identification data

5.1 specificity identification experiment

The rhNGF product of purifying is done immunoblotting and is identified, result and anti-hNGF antibody are positive.Show this product and anti-hNGF antibody generation immune response.

5.2N terminal Amino Acid Sequence Analysis

RhNGF to purifying carries out the N terminal Amino Acid Sequencing, and sequencing result shows that 15 aminoacid sequences of N end are:

Gly-Ala-Glu-Phe-Ser-Val-Cys-Asp-Ser-Val-Ser-Val-Trp-Val-Gly

Be that N terminal amino acid sequence and design meet.

6. determination of activity

Adopting the chick embryonic dorsal root ganglion method to measure, be specially take DMEM as mother liquor, in the substratum that contains 20% chicken plasma and 15% chick embryo extract, utilize esa brown chicken embryo lumbar vertebrae dorsal root ganglion, is under the stimulation of NGF of 30ng/ml at final concentration, cultivates 36h.After testing, its biologic activity of the rhNGF of purifying and mouse source Nerve Growth Factor Activity reach 10 at an order of magnitude ⁵More than the u/mg.

By above step, we have carried out the protein engineering transformation to hNGF, to obtaining the hNGF recombinant mutant (rhNGF) that drug effect is better, biological activity is more stable, are beneficial to study on mechanism and the clinical application of growth factor of human nerve.

Claims

1. the restructuring varient of the growth factor of human nerve among the present invention (rhNGF) is characterised in that the NGF peptide section N end of expression has lacked 10 amino acid of SSSHPIFHRG, many two amino acid of GA, see on the molecular weight and lacked 8 amino acid, begin to be same as the growth factor of human nerve normal sequence from EFSV.

2. growth factor of human nerve restructuring varient (rhNGF) the 1st amino acids is glycine among the present invention.

3. growth factor of human nerve restructuring varient (rhNGF) the 2nd amino acids is L-Ala among the present invention.

4. what the Asn-Gly peptide bond of cutting rhNGF fusion rotein was used among the present invention is the azanol patterning method.Oxammonium hydrochloride is the Ash-Gly peptide bond in the scinderin specifically, produces the N end and is the polypeptide of Gly.

5. the present invention adopts IPTG as the inductor abduction delivering, adopts carrying out ultrasonic bacteria breaking and osmose process to discharge most of solvable rhNGF fusion rotein, with Ni chelating chromatography column and CM-Sepharose chromatography column purifying rhNGF.

6. the growth factor of human nerve restructuring varient of the present invention's acquisition has pharmaceutical use, can develop the medicine of various formulations.Formulation refers to the pharmaceutical dosage forms such as powder pin, liquid drugs injection, spraying, lozenge, capsule, suppository.