CN106749605A

CN106749605A - A kind of growth factor of human nerve analog and preparation method thereof

Info

Publication number: CN106749605A
Application number: CN201611160674.3A
Authority: CN
Inventors: 汤华东; 万滔; 刘娜; 梅芸; 李玉薇; 陈亚
Original assignee: WUHAN HAITE BIOLOGICAL PHARMACEUTICAL CO Ltd
Current assignee: WUHAN HAITE BIOLOGICAL PHARMACEUTICAL CO Ltd
Priority date: 2016-12-15
Filing date: 2016-12-15
Publication date: 2017-05-31
Anticipated expiration: 2036-12-15
Also published as: CN106749605B

Abstract

The invention discloses a kind of growth factor of human nerve analog and preparation method thereof.The analog than growth factor of human nerve normal amino acid sequences N-terminal more than 1 glycine residue (G), its stability and expression quantity in Escherichia coli is set to be improved significantly, operational control is more beneficial for when from expressing fusion protein system simultaneously, so as to realize stabilization, solvable and high activity expression of the recombinant human nerve growth factor in escherichia expression system.

Description

A kind of growth factor of human nerve analog and preparation method thereof

Technical field

The present invention relates to biomedicine technical field, in particular to a kind of growth factor of human nerve analog and its Preparation method.

Background technology

Nerve growth factor (Nerve Growth Factor, NGF) be earliest find and most typical neurotrophy because Son, while being also one of most important bioactive molecule in nervous system.Its development to maincenter and peripheral neurons, point Change, grow, regeneration and the expression of functional characteristic are respectively provided with important regulating and controlling effect.Current NGF has been developed that into treatment periphery god Medicinal application through damaging is widely used in the nerves such as neurotrosis, hemiplegia, palsy, craniocerebral injury, child brain paralysis in clinic Property disease areas.But at present, the NGF of commercialization is mouse nerve growth factor (mNGF), potential immunogenicity risk.Naturally Growth factor of human nerve (hNGF) be distributed in brain, neuromere, iris, heart, spleen, placenta of human body etc. tissue, raw material sources It is limited, and wherein hNGF contents are low, isolate and purify extremely difficult, it is impossible to directly extract and obtain.

HNGF genes are located on No. 1 the short arm of a chromosome, complete 241 hNGF of amino acid composition of hNGF exons codings Precursor, commonly referred to preproNGF, the signal peptide (pre parts) of preproNGF is cut in endoplasmic reticulum, generation HNGF substances, i.e. proNGF (are made up of) 223 amino acid, are then transported in golgiosome, then gone through protease digestion Except leader peptide (pro parts), generation hNGF mature peptides (being made up of 120 amino acid) is transported to extracellular, performance physiology work( Energy.

In major expression systems, escherichia expression system is because its expression is high, low production cost, be easy to industrialization The advantages such as production turn into the first-selection of Prepare restructuring growth factor of human nerve.But the subject matter that presently, there are is：HNGF is difficult to The stabilization of recombinant human nerve growth factor, solvable and high activity expression are realized in escherichia expression system.

The content of the invention

It is an object of the invention to provide a kind of growth factor of human nerve analog, it is immunized with consistent with natural hNGF Activity and bioactivity, which obviate existing mouse nerve growth factor mNGF immunogenicity risk problems.

It is another object of the present invention to provide a kind of preparation method of growth factor of human nerve analog, weight is solved Group growth factor of human nerve is difficult to stable in escherichia expression system, solvable and high-activity expression technical barrier.

To realize the purpose of the present invention, using following technical scheme：

A kind of growth factor of human nerve analog (GhNGF), described growth factor of human nerve analog nerve containing people is raw Factor normal amino acid sequences long and its N-terminal increase a glycine residue.

Growth factor of human nerve analog as described above, it is preferable that the growth factor of human nerve normal amino acid sequence Be classified as natural growth factor of human nerve amino acid sequence, or with natural growth factor of human nerve amino acid sequence 95% with Upper homology and with the amino acid sequence of nerve growth factor BA.

Growth factor of human nerve analog as described above, it is preferable that the growth factor of human nerve analog is containing such as SEQ Amino acid sequence shown in ID NO.1.

Growth factor of human nerve analog as described above, it is preferable that the amino acid for encoding the SEQ ID NO.1 The corresponding nucleotide sequence of sequence is as shown in the 331st~693 in SEQ ID NO.2 or its complementary series.

A kind of preparation method of growth factor of human nerve analog, it is preferable that the growth factor of human nerve substance that will be transformed (proNGF) cDNA insertions prokaryotic expression carrier, is built into recombinant expression carrier, is expressed using escherichia expression system and prepared； Wherein, in the amino acid sequence such as SEQ ID NO.3 of growth factor of human nerve substance (proNGF) cDNA codings of the transformation 8th~231 shown sequence, or the sequence with its amino acid sequence homology more than 90%.

Preparation method as described above, it is preferable that the prokaryotic expression carrier is fusion protein expression vector, is that pMAL is carried Body, pGEX carriers or pET carriers.

Preparation method as described above, it is preferable that the pMAL carriers are pMAL-p5x, pMAL-p5E, pMAL-p5G, institute PET carriers are stated for pET39b or pET32a.

Preparation method as described above, it is preferable that described fusion protein expression vector have passed through transformation, and it eliminates load The fibrin ferment digestion recognition site of body itself.

Preparation method as described above, it is preferable that growth factor of human nerve substance (proNGF) cDNA of the transformation contains Sequence or its complementary series as shown in the 22nd~693 in SEQ ID NO.2.

Preparation method as described above, it is preferable that expression product prepared by the escherichia expression system expression is through pure After change, using fibrin ferment digestion, purifying again obtains growth factor of human nerve analog sterling.

The growth factor of human nerve analog that the present invention is provided, lives with the immunocompetence consistent with natural hNGF and biology Property, it is the recombinant human nerve growth factor (hNGF) prepared using genetic engineering recombinant protein technology.That replace tissue extraction Mouse nerve growth factor (mNGF), effectively prevent mNGF and there is a problem of immunogenicity risk, also solve natural The technical problem that can not directly obtain of growth factor of human nerve.

The present invention provides a kind of preparation method of growth factor of human nerve analog, in the normal amino of growth factor of human nerve Increase by 1 glycine residue (G) more the N-terminal of acid sequence, its stability and expression quantity in Escherichia coli is obtained substantially Improve, while operational control is more beneficial for when from expressing fusion protein system, so as to realize recombinant human nerve growth factor Stabilization, solvable and high activity expression in escherichia expression system.

Brief description of the drawings

Fig. 1 schemes for the SDS-PAGE of MBP-GhNGF amalgamation and expressions.

Fig. 2 is MBP-GhNGF amalgamation and expression Western Blot qualification figures.

Fig. 3 schemes for the SDS-PAGE of Trx-GhNGF amalgamation and expressions.

Fig. 4 is that different IPTG concentration are schemed in 28 DEG C of inductions on the SDS-PAGE of MBP-GhNGF amalgamation and expressions influence.

Fig. 5 is that different IPTG concentration are schemed in 25 DEG C of inductions on the SDS-PAGE of MBP-GhNGF amalgamation and expressions influence.

Specific embodiment

Inventor is by the further investigation to NGF molecular structures, it is proposed that a kind of growth factor of human nerve analog.Such Refer to that increased a glycine residue (G) in the N-terminal of growth factor of human nerve normal amino acid sequences like thing, be denoted as GhNGF, wherein, growth factor of human nerve normal amino acid sequences are natural growth factor of human nerve amino acid sequence, or are had Have with the natural homology of growth factor of human nerve amino acid sequence more than 95% and with nerve growth factor BA Amino acid sequence.By Optimal Expression, its stability and expression quantity in Escherichia coli is set to be improved significantly, while in choosing Operational control is more beneficial for during with expressing fusion protein system.

The present invention during growth factor of human nerve analog is prepared, to growth factor of human nerve substance (proNGF) Transformed, the amino acids (Lys of proNGF the 102nd for encoding it¹⁰²) it transform glycine (Gly as¹⁰²), and at the 103rd Amino acid (Arg¹⁰³) a glycine (Gly is inserted afterwards¹⁰⁴)；The amino acid sequence Gly encoded after transformation¹⁰²-Arg¹⁰³-↓Gly¹⁰⁴ (↓ be restriction enzyme site) can be recognized by fibrin ferment, and in Arg¹⁰³C-terminus cuts, produce many hNGF for glycine of N-terminal into Ripe peptide, i.e. growth factor of human nerve analog (GhNGF) prepared by the present invention.Identified through Western blotting (Western Blot) Being detected with bioactivity proves, GhNGF has the immunocompetence and bioactivity consistent with natural hNGF, and, GhNGF can Realize stable in escherichia expression system, solvable and high activity expression.Because GhNGF is the biology with new construction point Son, the expression of its solvable and high activity stable in escherichia expression system makes it possible its industrialization production, favorably In new drug development and clinical practice.

The present invention is illustrated below by specific embodiment, it should be noted that detailed description below is all exemplary, it is intended to Further instruction is provided to the present invention.

In this manual, unless specifically stated otherwise, technical term otherwise used is that those skilled in the art are normal Use term；The experimental technique of unreceipted actual conditions is routinely experimental technique in this specification；Examination used in this specification Test material and be commercially available purchase product unless otherwise instructed, the composition and compound method of various reagents and culture medium can be found in routine Operation in laboratory manual.

Embodiment 1：The acquisition of GhNGF genes

Growth factor of human nerve analog of the invention, is at natural growth factor of human nerve amino acid sequence and its N ends End increased a glycine residue, specific amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.1：

GSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHW NSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA。

In order to realize that recombinant human nerve growth factor is stable in escherichia expression system, solvable and high activity table Reach, inventor is with reference to Escherichia coli preference codon table, while the leading peptide-coding sequences of proNGF are transformed, by proNGF 102nd amino acids (Lys¹⁰²) it transform glycine (Gly as¹⁰²), and in the 103rd amino acids (Arg¹⁰³) afterwards insert one it is sweet Propylhomoserin (Gly¹⁰⁴), in addition herein in connection with the factor such as Codon degeneracy, G/C content, mRNA structures, the stability of transcription, design is suitable For the sequence of expression of recombinant proteins, the specific proNGF cDNA sequences as shown in SEQ ID NO.2, wherein, 5 ' hold and contain Contain the restriction enzyme sites of Hind III (AAGCTT) and terminator codon in the restriction enzyme sites of Ava I (CTCGGG), 3 ' ends In TAA, wherein SEQ ID NO.2 shown in the 331st~693 or its complementary series coding amino acid sequence correspond to SEQ ID Sequence shown in NO.1.

SEQ ID NO.2：(proNGF cDNA implementation sequences)

5’-CTCGGGGATGACGATGACAAAGAACCGCACTCCGAATCAAACGTTCCGGCTGGCCATACGATTCCG CAGGCGCACTGGACCAAACTGCAACATTCGCTGGATACCGCCCTGCGTCGCGCTCGTAGCGCACCGGCAGCAGCAAT TGCTGCGCGCGTGGCCGGTCAGACGCGTAATATCACCGTTGATCCGCGCCTGTTTAAAAAACGTCGCCTGCGTTCCC CGCGCGTCCTGTTCTCAACGCAGCCGCCGCGTGAAGCAGCAGACACCCAAGATCTGGACTTTGAAGTGGGCGGTGCT GCGCCGTTCAACCGTACCCATCGCTCTGGCCGTGGTAGCTCTAGTCATCCGATTTTTCACCGTGGCGAATTCAGTGT TTGCGATAGCGTGAGCGTTTGGGTCGGTGATAAAACCACGGCCACGGACATTAAAGGCAAAGAAGTGATGGTTCTGG GTGAAGTTAACATCAACAACAGCGTCTTCAAACAGTATTTCTTTGAAACCAAATGCCGCGATCCGAACCCGGTTGAC TCTGGCTGTCGTGGTATCGATTCTAAACACTGGAATAGTTACTGTACCACGACCCATACGTTTGTCAAAGCTCTGAC CATGGATGGCAAACAAGCCGCATGGCGCTTCATTCGTATCGACACCGCATGCGTCTGTGTGCTGAGCCGCAAAGCCG TGCGTCGCGCATAAAAGCTT-3’。

The sequence is synthesized by way of chemical synthesis.The amino acid sequence of above-mentioned proNGF cDNA sequences coding is such as Shown in SEQ ID NO.3, whereinGRGIt is fibrin ferment digestion recognition site.

SEQ ID NO.3：

LGDDDDKEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVL FSTQPPREAADTQDLDFEVGGAAPFNRTHRSGRGSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVN INNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA 。

The proNGF cDNA that the present invention is transformed, are the Codon degeneracy and e. coli codon according to amino acid Preferences, then consider the factors such as mRNA sequence structure, G/C content, the stability of cDNA sequence and its transcription, optimization, transformation Base sequence, is improved stability and expression quantity of the proNGF of its coding in Escherichia coli, the proNGF ammonia of coding Base acid sequence is equal to the 102nd amino acids (Lys of natural human proNGF amino acid sequences¹⁰²) it transform glycine as (Gly¹⁰²), and in the 103rd amino acids (Arg¹⁰³) a glycine (Gly is inserted afterwards¹⁰⁴) after sequence.The proNGF of coding Amino acid sequence sequence as shown in the 8th~231 in SEQ ID NO.3, or it is more than 90% with the amino acid sequence homology Sequence.

22nd~693 bit sequences of the proNGF cDNA of above-mentioned design as shown in SEQ ID NO.2 combines different fusions Protein expression vector, can realize purpose of the present invention albumen --- the expression of growth factor of human nerve analog.From difference Fusion protein expression vector need to design different pcr amplification primer things, introduce corresponding restriction enzyme site.Wherein be used for pET39b and The pcr amplification primer thing sequence of pET32a expression vectors is as follows：

SEQ ID NO.4：(forward primer, 5 ' ends introduce the restriction enzyme sites of Kpn IGGTACC)

5'-CGG GGTACC GATGACGATGACAAAGAACCGC-3'

SEQ ID NO.5：(reverse primer introduces the restriction enzyme sites of Hind IIIAAGCTTWith terminator codon TAA.)

5'-CCC AAGCTTTTATGCGCGACGCACG-3'。

Embodiment 2：The acquisition of the structure and engineering bacteria of GhNGF recombinant expression carriers

Expressing fusion protein is to add Special Proteins sequence in the N-terminal or C-terminal of destination protein, can with improve destination protein Dissolubility, promotes its correct folding, realizes its quick affinity purification, or realize its cell expression and localization.The Special Proteins sequence Be called fusion protein label (tag), conventional fusion protein label have maltose-binding protein (MBP), glutathione S- Transferase (GST), disulfide formation albumen (Dsb), thioredoxin (Trx) and His-tag, S-tag etc..Using which The carrier of expression is referred to as fusion protein expression vector, such as pMAL carriers are using MBP as fusion protein label, pGEX carriers Using GST as fusion protein label.

The present invention uses prokaryotic expression carrier, is capable of achieving the purpose of the present invention, preferably fusion protein expression vector；Specifically PMAL carriers, pGEX carriers or pET carriers can be used；It is further preferable that for pMAL-p5x, pMAL-p5E, pMAL-p5G or PET39b or pET32a；Most preferably, being pET39b or pET32a.It is listed below to illustrate：

2.1 with pMAL-p5x as fusion protein expression vector, builds GhNGF recombinant expression carriers and amalgamation and expression engineering Bacterium.

By artificial synthesized proNGF cDNA sequences (SEQ ID NO.2), with passing through after Ava I and the double digestions of Hind III T4DNA ligases are connected to same through on the pMAL-p5x expression vectors of double digestion, building pMAL-p5x-GhNGF recombination expressions Carrier, converts escherichia coli cloning bacterial strain Top10,37 DEG C of culture sieves on the LB culture mediums containing 50 μ g/ml ampicillins Select recon, through PCR and digestion identification it is correct after, sequence verification its correct sequence.

Extraction identifies that correct recon, for pMAL-p5x-GhNGF recombinant expression carriers, is transformed into E. coli competent In expression bacterial strain BL21 (DE3), on the LB culture mediums containing 50 μ g/ml ampicillins (Amp), 37 DEG C of culture screening restructuring Son, the recon of acquisition is the GhNGF engineering bacterias of MBP amalgamation and expressions, is named as MBL-GhNGF.It is stored in 15% sweet In oil, freeze in -80 DEG C of refrigerators.Wherein, the fusion protein that MBL-GhNGF engineering bacterium expressions are produced should be combined containing maltose Albumen (MBP) label, the pro parts of hNGF substances and GhNGF sequences, theoretical molecular are about 70kDa.

The MBL-GhNGF engineering bacterias that glycerol tube freezes are taken, is inoculated in containing fresh LB resistance cultures base (containing 50 μ by 0.2% G/ml Amp) test tube in, shaken cultivation overnight, activated strains；It is forwarded to again in the LB culture mediums of 100mL, 37 DEG C of vibration trainings About 3h is supported, its OD600 value is surveyed at 0.6~0.8,0.5mM isopropyl-β-D-thiogalactosides (IPTG) is added, in 37 DEG C Induced expression 4h, is collected by centrifugation thalline；Thalline weight in wet base is weighed, (25mM Glycine-NaOHs are buffered by thalline and broken bacterium solution Liquid, pH10.0) weight ratio adds broken bacterium solution for 1: 15 ratio, after carrying out ultrasonic bacteria breaking, bacteria break supernatant and precipitation is carried out into SDS- PAGE is detected.Result is as shown in figure 1, wherein M is albumen Marker；1 be 0.5mM IPTG, 37 DEG C induction 4h bacteria break supernatant；2 For the broken bacterium of 0.5mM IPTG, 37 DEG C of induction 4h is precipitated.There is the fusion protein of molecular weight about 70kDa in result explanation bacteria break supernatant Expression.

The fusion protein of the molecular weight of above-mentioned expression about 70kDa is used into proNGF monoclonal antibodies (Abcam Products, goods respectively Number：Ab68151, similarly hereinafter.) and NGF monoclonal antibodies (Abcam Products, article No.：Ab52918, similarly hereinafter.) carry out Western Immuno print Mark (Western Blot) is identified, as a result as shown in Fig. 2 wherein Fig. 2A is to hybridize to identify using proNGF monoclonal antibodies；Fig. 2 B are to adopt Hybridized with NGF monoclonal antibodies and identified；1 is BL21 (DE3) the zero load bacterium without induction；2 is not induce MBL-GhNGF engineering bacteria thalline complete Albumen；3 is 0.2mM IPTG, the broken bacterium of 28 DEG C of induction 4h is precipitated；4 be 0.2mM IPTG, 28 DEG C induction 4h bacteria break supernatant.Knot Fruit shows that the fusion protein of the 70kDa in bacteria break supernatant has proNGF and NGF immunocompetences.

2.2 with pET39b as fusion protein expression vector, builds GhNGF recombinant expression carriers and amalgamation and expression engineering bacteria.

It is template with the 2.1 recombinant vector pMAL-p5x-GhNGF for building, using SEQ ID NO.4 and SEQ ID NO.5 Shown sequence is primer, expands proNGF cDNA sequences, its sequence 5 ' is held with the restriction enzyme sites of Kpn I, 3 ' ends Sequence is constant, still contains the restriction enzyme sites of Hind III and terminator codon TAA.

Specifically, PCR reaction systems are 50 μ l, the μ l of template 1 of wherein pMAL-p5x-GhNGF, with embodiment 1 Forward and reverse primer (10 μM) of the sequence as shown in SEQ ID NO.4 and SEQ ID NO.5 each 1 μ l, dNTPs 3 μ l, 5 × 10 μ l, Taq archaeal dna polymerases of Buffer 1 μ l, ddH₂O33μl.Amplification reaction condition is 95 DEG C of predegeneration 5min；95 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 circulations；72 DEG C of extension 10min after last circulation.Use 1.2% agar Pcr amplification product is reclaimed in sugared gel electrophoresis, after the pcr amplification product after recovery is through Kpn I and the double digestions of Hind III, by T4DNA Ligase is connected on the same pET39b expression vectors through Kpn I and the double digestions of Hind III, builds pET39b-GhNGF restructuring tables Up to carrier, escherichia coli cloning bacterial strain DH5 α are converted, 37 DEG C of trainings on the LB culture mediums containing 50 μ g/ml kanamycins (Kan) Support screening recon, through PCR and digestion identification it is correct after, sequence verification its correct sequence.The correct restructuring of identification is extracted to carry The recombinant plasmid of body pET39b-GhNGF, conversion E. coli competent expresses bacterial strain BL21 (DE3), is containing 50 μ g/ml cards 37 DEG C of culture screening recons on the LB culture mediums of that mycin, the recon of acquisition is the GhNGF engineerings of DsbA amalgamation and expressions Bacterium, is named as D39BL-GhNGF.Wherein, the fusion protein that D39BL-GhNGF engineering bacterium expressions are produced should contain disulfide formation Albumen (DsbA) label, the pro parts of hNGF substances and GhNGF sequences, theoretical molecular are about 53kDa.

The D39BL-GhNGF engineering bacterias that glycerol tube freezes are taken, is inoculated in by 0.2% and (contained containing fresh LB resistance cultures base 50 μ g/ml Kan) test tube in, shaken cultivation overnight, activated strains；It is forwarded to again in the LB culture mediums of 100mL, 37 DEG C of vibrations Culture about 3h, surveys its OD600 value at 0.6~0.8, adds 0.2mM IPTG, in 25 DEG C of induced expression 4h, bacterium is collected by centrifugation Body；Thalline weight in wet base is weighed, by thalline and broken bacterium solution (20mM Tris-HCl buffer solutions, pH8.0) weight ratio for 1: 15 ratio adds Enter brokenly bacterium solution, after carrying out ultrasonic bacteria breaking, bacteria break supernatant and precipitation are carried out into SDS-PAGE detections and Western Blot identifications.Result is said There is the expressing fusion protein of molecular weight about 53kDa in bright bacteria break supernatant；And the fusion protein has the immune work of proNGF and NGF Property.

2.3 with pET32a as fusion protein expression vector, builds GhNGF recombinant expression carriers and amalgamation and expression engineering bacteria.

To improve the fusion protein DsbA-proNGF of expression by the efficiency of fibrin ferment digestion, can be by PCR rite-directed mutagenesises Method eliminates the coded sequence of fibrin ferment digestion recognition site (LVPRGS) that carrier is carried in itself, such as by the amino acid sequence from LVPRGS sports LVPKGS.

2.3.1 pET32a is transformed, the coded sequence of its fibrin ferment digestion recognition site (LVPRGS) for carrying is eliminated.

Synthesize following primer (SEQ ID NO.6, SEQ ID NO.7), with rite-directed mutagenesis fibrin ferment digestion recognition site (LVPRGS) coded sequence, will wherein R (arginine) sequence encoding mutant for K (lysine) coded sequence.

SEQ ID NO.6：(just strand primer, whereinAAAIt is lysine coded sequence)

5’-AAAAGGTTCTGGTATGAAAGAAACCGCTGC-3’

SEQ ID NO.7：(antisense strand primer, whereinTTTIt is lysine coded sequence)

5’-TCATACCAGAACCTTTTGGCACCAGACCAG-3’

With pET32a carriers as template, using sequence shown in SEQ ID NO.6 and SEQ ID NO.7 for primer is expanded Increase.PCR system is 30 μ l, wherein template plasmid 1 the μ l, above-mentioned each 0.8 μ l, dNTPs (10 μ of primer (10 μM) of pET32a carriers M) the μ l of 0.6 6 μ l, Phusion high-fidelity DNA polymerase of μ l, 5 × Buffer 0.3, add ddH₂The μ of O to 30 l.Amplified reaction bar Part is 98 DEG C of 1min；98 DEG C of 5s, 61 DEG C of 30s, 72 DEG C of 3min, 32 circulations；72℃10min.Use 1.2% agarose gel electrophoresis Pcr amplification product is reclaimed, after the digestions template plasmids of Dpn I, conversion escherichia coli cloning bacterial strain Top10, selected clone bacterium Fall, sequence verification its correct sequence.Plasmid contained by correct clone bacterium is sequenced is the pET32a carriers of transformation, is denoted as pET32a-deT。

2.3.2 pET32a-deT-GhNGF recombinant expression carriers and engineering bacteria are built

The product for using SEQ ID NO.4 and SEQ ID NO.5 to be expanded for primer in 2.2 is taken, with Kpn I and Hind III couple After digestion, it is connected on the same pET32a-deT expression vectors through Kpn I and the double digestions of Hind III by T4DNA ligases, structure PET32a-deT-GhNGF recombinant expression carriers are built, escherichia coli cloning bacterial strain Top10 is converted, blue or green containing 50 μ g/ml ammonia benzyls 37 DEG C of culture screening recons on the LB culture mediums of mycin, recon through PCR and digestion identification it is correct after, sequence verification its sequence Correctness.

The recombinant plasmid of the correct recombinant vector pET32a-deT-GhNGF of identification is extracted, E. coli competent table is converted Up to bacterial strain Origami B (DE3), 37 DEG C of culture screening recons, obtain on the LB culture mediums containing 50 μ g/ml ampicillins The recon for obtaining is the GhNGF engineering bacterias of TrxA amalgamation and expressions, is named as T32deTOB-GhNGF.It is stored in 15% sweet In oil, freeze in -80 DEG C of refrigerators.Wherein, the fusion protein that T32deTOB-GhNGF engineering bacterium expressions are produced should be containing sulphur oxygen also Albumen (TrxA) label, the pro parts of hNGF substances and GhNGF sequences, theoretical molecular are about 42kDa.

The T32deTOB-GhNGF engineering bacterias after activation are taken, 1: 100 ratio is seeded to 100ml LB cultures by volume In base, when OD600 reaches 0.6, the IPTG, 25 DEG C of induced expression 4h of final concentration of 0.5mM are added；Control is not added with IPTG, together Sample CMC model.Thalline is collected by centrifugation, the PBS of 15ml, carrying out ultrasonic bacteria breaking is added, condition is ultrasound 2s, interval 3s, 80% power； Broken bacterium separates bacteria break supernatant and precipitation after 4 DEG C of 12000rpm centrifugation 15min, and the PBS of precipitation equal volume is resuspended.Respectively Bacterium solution, bacteria break supernatant and the precipitation re-suspension liquid of 20 μ l are taken, 5 μ l 5 × loading Buffer are added, mixed, in 100 DEG C of boiling water baths Denaturation 5min, respectively takes 10 μ l, row SDS-PAGE detections.Result is as shown in figure 3, wherein 1 is not induce whole bacterial protein；2 is not lure Lead brokenly bacterium precipitation；3 is not induce bacteria break supernatant；4 is the whole bacterial protein of induced expression；5 precipitate for the broken bacterium of induced expression；6 are The bacteria break supernatant of induced expression.Result shows the target protein for having molecular size range about 42kDa in bacteria break supernatant.

The fusion protein of the molecular weight of above-mentioned expression about 42kDa is carried out with proNGF monoclonal antibodies and NGF monoclonal antibodies respectively Western Blot identify that as a result showing the fusion protein of 42kDa has proNGF and NGF immunocompetences.

Embodiment 3：The expression and purifying of GhNGF

3.1MBP amalgamation and expressions GhNGF and purifying

3.1.1MBP the condition optimizing of amalgamation and expression GhNGF

The MBL-GhNGF engineering bacterias that will be activated in embodiment 2.1, are inoculated in LB culture mediums, and 37 DEG C of shaken cultivations are extremely When OD600 is 0.6, respectively at 28 DEG C of inducing temperature, IPTG concentration is that 0mM, 0.05mM, 0.2mM induce 4h；Inducing temperature 25 DEG C, IPTG concentration is that 0mM, 0.1mM, 0.2mM, 0.5mM induce 4h, and thalline is collected by centrifugation, after carrying out ultrasonic bacteria breaking, by bacteria break supernatant and Precipitation carries out SDS-PAGE detections.Experimental result as shown in Figure 4, Figure 5, wherein, in Fig. 41 be albumen Marker；2 is not induce Whole bacterial protein；3 is the bacteria break supernatant of 0.05mM IPTG induced expressions；4 precipitate for the broken bacterium of 0.05mM IPTG induced expressions；5 It is the bacteria break supernatant of 0.2mM IPTG induced expressions；6 precipitate for the broken bacterium of 0.2mM IPTG induced expressions；In Fig. 5,1 is albumen Marker；2 is not induce whole bacterial protein；3 is the bacteria break supernatant of 0.05mM IPTG induced expressions；4 induce for 0.05mM IPTG The broken bacterium precipitation of expression；5 is the bacteria break supernatant of 0.1mM IPTG induced expressions；6 sink for the broken bacterium of 0.1mM IPTG induced expressions Form sediment.7 is the bacteria break supernatant of 0.2mM IPTG induced expressions；8 precipitate for the broken bacterium of 0.2mM IPTG induced expressions；9 is 0.5mM The bacteria break supernatant of IPTG induced expressions；10 precipitate for the broken bacterium of 0.5mM IPTG induced expressions.

Result shows, is 25 DEG C in inducing temperature, and during IPTG concentration 0.2mM induction 4h, soluble protein expression quantity is relative It is more.

3.1.2MBP the purifying of amalgamation and expression GhNGF

The expression for recombinating GhNGF albumen is completed using the MBL-GhNGF engineering bacterias of the structure of embodiment 2.1.After fermentation Zymotic fluid is collected by centrifugation thalline, and the broken bacterium of pressure homogenate collects Dextrin Sepharose HP chromatographic columns on supernatant, with containing malt The buffer solution (200mM maltose -20mM trishydroxymethylaminomethanes (Tris), pH8.0) of sugar washes lower fusion protein, adds blood coagulation The digestion 2 hours of 25 DEG C of enzyme, then by Dextrin Sepharose HP chromatographic columns, except foreigh protein removing, (total length of non-digestion is merged Albumen and fusion protein label-hNGF leader peptides (MBP-pro peptide)), the GhNGF for cutting is through SP Sepharose FF Chromatography obtains GhNGF of the purity more than 95%.

3.2DsbA amalgamation and expressions GhNGF and purifying

The expression for recombinating GhNGF albumen is completed using the D39BL-GhNGF engineering bacterias of the structure of embodiment 2.2.Fermentation After fermentation liquid is collected by centrifugation thalline, and the broken bacterium of pressure homogenate is collected Ni-Sepharose 6FF chromatographic columns on supernatant, uses 50mM- 200mM imidazoles (Imidazole) gradient elution collects fusion protein, through the digestion 2 hours of 25 DEG C of fibrin ferment, then by Ni- Sepharose 6FF chromatographic columns remove foreigh protein removing (the total length fusion protein and fusion protein label-hNGF leader peptides of non-digestion (Dsb-pro peptide)), the GhNGF for cutting obtains GhNGF of the purity more than 95% through SP Sepharose FF chromatographies.

3.3TrxA amalgamation and expressions GhNGF and purifying

The expression for recombinating GhNGF albumen is completed using the T32deTOB-GhNGF engineering bacterias of the structure of embodiment 2.3.Hair Ferment after fermentation liquid is collected by centrifugation thalline, and pressure homogenate is broken bacterium, collects supernatant, with the purifying of 3.2 methods, obtains purity and is more than 95% GhNGF。

The GhNGF that above-mentioned three kinds of methods obtain after purification carries out Western with proNGF monoclonal antibodies and NGF monoclonal antibodies respectively Blot identifies that above-mentioned GhNGF after purification is respectively provided with proNGF and NGF immunocompetences again.

Embodiment 4：The identification of GhNGF

4.1N terminal amino acid sequences are detected

The GhNGF that in embodiment 3 prepared by three kinds of expression system expression, the sequencing result of 15 amino acid of N-terminal is： GSSSHPIFHRGEFSV, it is consistent with design load.

4.2 bioactivity are detected

With the mouse nerve growth factor reference material of Chinese pharmaceutical biological product calibrating research institute distribution as reference, use《In State's pharmacopeia》Three general rules 3530 of version in 2015 " TF-1 cells/MTS colorimetric methods " detected, three kinds of expression systems in embodiment 3 The specific activity for expressing the GhNGF for preparing is respectively 6.37 × 10⁵U/mg、5.71×10⁵U/mg and 7.12 × 10⁵U/mg。

Above example shows that GhNGF can realize stable in escherichia expression system, solvable and high activity table Reach, so as to be conducive to its industrialization production to prepare, and new drug development and clinical practice.

It should be pointed out that the foregoing is only the preferred embodiments of the present invention, come for the those of ordinary skill in this technology Say, on the premise of core technical features of the invention are not departed from, some improvements and modifications can also be made, these are retouched and change Entering should also belong to scope of patent protection of the invention.

SEQUENCE LISTING

<110>Wuhan Haite Bio-pharmaceutical Co., Ltd

<120>A kind of growth factor of human nerve analog and preparation method thereof

<130>

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 121

<212> PRT

<213>Artificial sequence

<400> 1

Gly Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys

1 5 10 15

Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile

20 25 30

Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser

35 40 45

Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro

50 55 60

Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr

65 70 75 80

Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys

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Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val

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Leu Ser Arg Lys Ala Val Arg Arg Ala

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<212> DNA

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ctcggggatg acgatgacaa agaaccgcac tccgaatcaa acgttccggc tggccatacg 60

attccgcagg cgcactggac caaactgcaa cattcgctgg ataccgccct gcgtcgcgct 120

cgtagcgcac cggcagcagc aattgctgcg cgcgtggccg gtcagacgcg taatatcacc 180

gttgatccgc gcctgtttaa aaaacgtcgc ctgcgttccc cgcgcgtcct gttctcaacg 240

cagccgccgc gtgaagcagc agacacccaa gatctggact ttgaagtggg cggtgctgcg 300

ccgttcaacc gtacccatcg ctctggccgt ggtagctcta gtcatccgat ttttcaccgt 360

ggcgaattca gtgtttgcga tagcgtgagc gtttgggtcg gtgataaaac cacggccacg 420

gacattaaag gcaaagaagt gatggttctg ggtgaagtta acatcaacaa cagcgtcttc 480

aaacagtatt tctttgaaac caaatgccgc gatccgaacc cggttgactc tggctgtcgt 540

ggtatcgatt ctaaacactg gaatagttac tgtaccacga cccatacgtt tgtcaaagct 600

ctgaccatgg atggcaaaca agccgcatgg cgcttcattc gtatcgacac cgcatgcgtc 660

tgtgtgctga gccgcaaagc cgtgcgtcgc gcataaaagc tt 702

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Leu Gly Asp Asp Asp Asp Lys Glu Pro His Ser Glu Ser Asn Val Pro

1 5 10 15

Ala Gly His Thr Ile Pro Gln Ala His Trp Thr Lys Leu Gln His Ser

20 25 30

Leu Asp Thr Ala Leu Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile

35 40 45

Ala Ala Arg Val Ala Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg

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Leu Phe Lys Lys Arg Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr

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Gln Pro Pro Arg Glu Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val

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Gly Gly Ala Ala Pro Phe Asn Arg Thr His Arg Ser Gly Arg Gly Ser

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Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp Ser

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Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly

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Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val Phe

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Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val Asp

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Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys Thr

180 185 190

Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln Ala

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Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu Ser

210 215 220

Arg Lys Ala Val Arg Arg Ala

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cccaagcttt tatgcgcgac gcacg 25

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<212> DNA

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aaaaggttct ggtatgaaag aaaccgctgc 30

<210> 7

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<213>Artificial sequence

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tcataccaga accttttggc accagaccag 30

Claims

1. a kind of growth factor of human nerve analog, it is characterised in that described growth factor of human nerve analog is containing people's nerve Growth factor normal amino acid sequences and its N-terminal increase a glycine residue.

2. growth factor of human nerve analog as claimed in claim 1, it is characterised in that the growth factor of human nerve is normal Amino acid sequence be natural growth factor of human nerve amino acid sequence, or with natural growth factor of human nerve amino acid sequence The homology of row more than 95% and with the amino acid sequence of nerve growth factor BA.

3. growth factor of human nerve analog as claimed in claim 1, it is characterised in that the growth factor of human nerve is similar to Thing is containing the amino acid sequence as shown in SEQ ID NO.1.

4. growth factor of human nerve analog as claimed in claim 3, it is characterised in that for encoding the SEQ ID The corresponding nucleotide sequence of amino acid sequence of NO.1 is as shown in the 331st~693 in SEQ ID NO.2 or its complementary series.

5. a kind of preparation method of growth factor of human nerve analog, it is characterised in that the growth factor of human nerve that will be transformed is former Body cDNA inserts prokaryotic expression carrier, is built into recombinant expression carrier, is expressed using escherichia expression system and prepared；Wherein, The amino acid sequence of the growth factor of human nerve substance cDNA codings of the transformation is as shown in the 8th~231 in SEQ ID NO.3 Sequence, or the sequence with its amino acid sequence homology more than 90%.

6. preparation method as claimed in claim 5, it is characterised in that the prokaryotic expression carrier is that expressing fusion protein is carried Body, is pMAL carriers, pGEX carriers or pET carriers.

7. preparation method as claimed in claim 6, it is characterised in that the pMAL carriers be pMAL-p5x, pMAL-p5E, PMAL-p5G, the pET carriers are pET39b or pET32a.

8. preparation method as claimed in claim 6, it is characterised in that described fusion protein expression vector have passed through transformation, Its fibrin ferment digestion recognition site for eliminating carrier itself.

9. preparation method as claimed in claim 5, it is characterised in that the growth factor of human nerve substance cDNA of the transformation contains Sequence or its complementary series as shown in the 22nd~693 in SEQ ID NO.2.

10. the preparation method as described in claim 5-9 is any, it is characterised in that the escherichia expression system expression system After standby expression product is purified, using fibrin ferment digestion, purifying again obtains growth factor of human nerve analog sterling.