CN102911265B - Recombination variant of human nerve growth factors and preparation method thereof - Google Patents

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 to regulating effect in 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.With genetically engineered Restruction growth factor of human nerve, it is unique approach.RhNGF has important clinical value, will fill up the blank of international neural repair medicine.China has nerve injury patients up to a million every year, and market outlook are considerable.

We have invented a kind of hNGF restructuring varient of transforming acquisition by protein engineering 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 start to have lacked S Serine from N, S Serine, S Serine, H Histidine, P proline(Pro), I Isoleucine, F phenylalanine, H Histidine, 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, F phenylalanine, S Serine, V α-amino-isovaleric acid and backward amino acid start to meet hNGF normal sequence, from bulk molecule amount, 8 amino acid have been lacked.The protein engineering transformation that we carry out hNGF by above-mentioned 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.

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 according to the gene order of the hNGF of our molecular designing and reference report.The sequence that 5 ' end of the hNGF gene order of our design contains Kpn1 restriction enzyme site and coding azanol cleavage site, 3 ' end contains Xba1 restriction enzyme site and termination codon TGA.Synthetic gene implementation sequence is shown in Fig. 1.

Fig. 1 .rhNGF synthetic gene implementation sequence

2. gene sequencing

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

Order-checking collection of illustrative plates is used tricks to calculate machine-readable order, obtain cDNA sequence and translate into corresponding amino acid, cDNA and the consensus amino acid sequence of the hNGF of our design of result.Show that the cDNA that we clone the rhNGF obtaining is correct.

3. the structure of expression plasmid

The cDNA of rhNGF is inserted in pBV220 and directly expressed, and expression product forms insoluble occlusion body, need to obtain activated rhNGF through denature and renature.Because rhNGF is containing 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 rhNGF and escherichia coli thioredoxin thioredoxin, and thioredoxin can guide albumen thereafter correctly folding, is solubility expression, tool natural biological is learned active, has avoided a difficult problem for sex change renaturation.

What cutting method was used is oxammonium hydrochloride patterning method.Oxammonium hydrochloride is the Asn-Gly peptide bond in scinderin specifically, and producing N end is the polypeptide of Gly.We find in rhNGF polypeptide containing 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 is with discharging rhNGF after azanol cutting, and its initial amino acid is Gly, and aminoacid sequence is after this consistent with rhNGF sequence.Gly is similar to Met character, and being placed on N end does not all affect the activity of albumen, moreover the N terminal amino acid of hNGF is inessential to activity, and first 8 amino acid of its N end will be hydrolyzed with practical function in human body.

Azanol is the eubolism product in human body, and oxammonium hydrochloride cost is low, and cleavage specificity is good, and cutting efficiency is high, is applicable 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 Amp resistant gene.We insert synthetic rhNGF cDNA between the Kpn1-Xba1 of above-mentioned plasmid by the fragment after Kpn1-Xba1 double digestion, are built into expression plasmid pTHNGF.

Host Strains is selected e. coli jm109 (purchased 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, forms engineering bacteria pTHNGF/JM109.Be stored in 15% glycerine, be frozen in-70 ℃, be original species word bank.

The calibrating of 4.rhNGF engineering bacteria

4.1 ne ar, cultural characteristic, physio-biochemical characteristics

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

4.1.1 ne ar

With after seed liquor coating slide, gramstaining 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

With seed liquor line, LBA is dull and stereotyped, cultivates 20 hours for 37 ℃, and naked eyes are visible: bacterium colony is circular, the smooth of the edge, and bacterium colony is full, and surface wettability, smooth, is creamy white, and 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 features

Unconverted Host Strains BL21 grows on LB substratum, and (Amp:0.1mg/ml) do not grow on 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.With KpnI-XbaI and KpnI-PstI, all can cut out the small band of 370bp, can cut out the small band of about 340bp with EcoRI-PstI, enzyme is cut qualification result and is all met plasmid design, illustrates that plasmid pTHNGF builds correct.

The expression of 4.4 engineering bacterias and the acquisition of expression product

PTHNGF/JM109 seed liquor is inoculated in LBA substratum with 1% ratio, and 37 ℃ grow to OD ₆₀₀be about 0.5, add IPTG to 1mM as inductor, induce 5 hours for 37 ℃, results thalline, after washing, sampling cracking, carries out SDS-PAGE electrophoretic analysis.Thalline after visible induction has more a dense protein band at 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 the 10-20% left and right of bacterial protein.

The thalline of expressing is carried out to carrying out ultrasonic bacteria breaking, and high speed centrifugation.Precipitation and supernatant are carried out to SDS-PAGE electrophoresis.The rhNGF fusion rotein of visible expression concentrates in supernatant, with soluble form, expresses, and tool natural radioactivity, does not need through denature and renature.

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

5. expression product structural identification data

5.1 specificity identification experiments

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

The rhNGF of purifying is carried out to 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

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

By above step, we have carried out 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 (4)

1. a growth factor of human nerve restructuring varient (rhNGF), is characterized in that being comprised of aminoacid sequence GAEFSV CDSVSVWVG DKTTATDIK GKEVMVLGE VNINNSVFK EYFFETKCR DPNPVDSGC RGIDSKHWN SYCTTTHTF VKALTMDGK EAAWRFIRI DTACVCVLS RKAVR.

2. the preparation method of growth factor of human nerve restructuring varient according to claim 1, it is characterized in that, select plasmid pThioHis A as expression vector, it contains promotor Ptrc promotor, thioredoxin leading peptide and terminator aspA terminator, and Amp resistant gene; Synthetic rhNGF cDNA is inserted between the Kpnl-Xbal of above-mentioned plasmid by the fragment after Kpnl-Xbal double digestion, be built into expression plasmid pTHNGF.

3. the preparation method of growth factor of human nerve restructuring varient according to claim 1, it is characterized in that, selected Asn-Gly is tie point and thioredoxin amalgamation and expression rhNGF, with the oxammonium hydrochloride Asn-Gly peptide bond in cleavage of fusion proteins specifically, producing N end is the rhNGF polypeptide of Gly again.

4. the preparation method of growth factor of human nerve restructuring varient according to claim 1, it is characterized in that, purifying is by osmotic pressure method, the solvable rhNGF fusion rotein major part in Bacillus coli cells to be discharged in damping fluid, after azanol cutting, with Ni chelating chromatography column and CM-Sepharose chromatography column, purify and obtain.