CN108034678A - A kind of expression vector, system and method for expressing recombinant human nerve growth factor - Google Patents

Abstract

The present invention relates to the eukaryotic expression system and method that one kind prepares growth factor of human nerve (rhNGF) albumen, use recombined lentivirus vector and express the Chinese hamster ovary celI of rhNGF, by a series of purification procedures, the growth factor of human nerve albumen of high activity high-purity is finally obtained, there are market prospects.

Description

A kind of expression vector, system and method for expressing recombinant human nerve growth factor

Technical field

The present invention relates to a kind of eukaryotic cell expression system of expression growth factor of human nerve (rhNGF) albumen；

The invention further relates to the preparation method of recombinant human nerve growth factor.

Background technology

Nerve growth factor (NGF) is found earliest in neurotrophic factor, and research is the most thorough at present, has god A kind of nerve growth regulatory factor through first nutrition and rush enation double biological function, is that nervous system is most important One of bioactive molecule, its development, differentiation, growth, regeneration and expression of functional characteristic to maincenter is respectively provided with important Regulating and controlling effect, the Regeneration and Repair for exchanging the growth of ganglion neuron, development, differentiation, survival and injured nerve have important theory And clinical meaning.Current basic research shows that NGF can be applied to neurotoxic, peripheral nerve injury, Diabetic Peripheral god Through lesion, senile dementia, Parkinson's disease, facial neuritis, and neurotrosis (including spinal cord injury, high paraplegia, severed finger Replant, the damage of the maincenter such as brain damage and Peripheral Nervous) etc. the nervous system disease, be that currently the only can be applied to clinic Nervous system rho factor.

NGF mRNA, mRNA are generated after ngf gene transcription and synthesizes the precursor protein of NGF after translating first.Precursor protein has Two kinds, a kind of is long precursor protein, and a kind of is short precursor protein.The mRNA of short precursor protein is encoded positioned at single outer In aobvious son, so it is two kinds that NGF, which is divided to, i.e. 7SNGF and 2.5SNGF, usually signified NGF is 2.5SNGF.2.5SNGF is by 118 The dimer of a amino acid composition, the bioactivity of whole NGF rely on β subunits entirely, its sedimentation coefficient is 2.5S, also known as 2.5SNGF.Complete hNGF molecules are combined into 2 β of α by 1 β subunit, 2 alpha subunits and 2 γ subunits with non-covalent bond The form of γ 2, further includes 2 zinc fingers in molecule, to increase the stability of compound.β sub-unit molecules amount is 26 518, its pI It is 9.3, dimer is formed by non-covalent chain combination.Wherein, disulfide bond is connected between 6 half Guang amino acid residues, this A little disulfide bond are particularly significant to the bioactivity for maintaining NGF, the active total loss if destroying.

Content is little in human body by hNGF, and natural origin is hardly possible.Therefore, restructuring is produced with the method for genetic engineering Growth factor of human nerve (rhNGF) is uniquely to select.The commercialized product of nerve growth factor is mainly derived from mouse at present Salivary gland extracts, and male mice salivary gland is the main donor for purifying NGF and analyzing its bioactivity.The injection that China develops It is the kind biological product New Drug Certificate that acquisition State Food and Drug Administration (SFDA) issues with mouse nerve growth factor. Chinese Pharmacopoeia 2015 editions provides mouse source NGF its protein content is not less than 0.1mg/ml, and specific activity is not less than 5*10⁵AU/mg (Chinese Pharmacopoeia Commission compiles, Pharmacopoeia of People's Republic of China 2015 editions).The limited public affairs of road bioengineering of Xiamen Beijing University in 2001 The NGF of department is granted, becomes a NGF medicine that beat the world.Up to now, it is domestic to have had the granted NGF of multiple producers, Er Qiecheng For one of best-selling medicine, according to IMS data, for China's NGF sales volumes in 2016 up to 11,000,000, gross sales amount is beautiful up to 2.15 hundred million First (pressing producer price).

On July 6th, 2017, EMA have approved the recombinant human nerve growth factor (cenegermin) of Italian Domp é companies Eye drops is treated for middle severe neurological trophic keratitis (NK), and neurotrophic keratitis is a kind of rare ophthalmology disease Disease, therapeutic scheme lack, weak curative effect, because incidence less than 5/10000ths, cenegermin is granted by Orphan drug qualification by EMA. Its phase ii clinical trial the results show that this product all shows extraordinary curative effect, the complete cure rate for the treatment of group is significantly higher than Placebo.This product good security, main adverse reaction are ophthalmodynia, shed tears.Oxervate is given birth to by recombinant DNA technology Production, avoids the infection risk of animal sources NGF viruses, and quality is more controllable.Oxervate is with eye drops (20 μ g/mL) Form is applied to neurotrophic keratitis patient, easy to operate, and auxiliary recovers the normal healing processes of eye, repairing corneal disturbance Work well.The said firm announces that Oxervate will log in European market within year in 2017, and prepares to FDA and PMDA applications NDA。

So far, male mice salivary gland is the main donor for purifying NGF and analyzing its bioactivity, but mouse source NGF has following significant drawback：1. need, using a large amount of mouse separation submandibular organization, to run counter to animal welfare principle；② Product quality is difficult to ensure that, also, NGF contents are low and are difficult to avoid that the pollution of heterologous protein；3. organization material pretreatment with And purifying process is all complex；4. more importantly mouse NGF amino acid composition still has with space structure compared with people NGF Notable difference, long-time service have potential danger, to upgrade there is an urgent need for developing recombined human NGF, substitute existing product.

The more traditional mouse submandibular gland extraction process of production of genetic recombination NGF has a clear superiority.Utilize extracorporeal recombination β-NGF are synthesized, although can be expressed in prokaryotic, because the polypeptide of synthesis cannot be folded so that the relatively low (Liu Dong of activity The clone of the hNGF-β gene subunits cDNA such as sea and the expression in Escherichia coli, Chinese Journal of Pathophysiology, 2002, 5:39-42).Expressed in eukaryotic, be more advantageous to obtaining the higher and closer β-NGF albumen synthesized naturally of activity, and Because its molecule small (molecular weight is less than 40kD) is easy to pass through blood-brain barrier (Han Xiaofeng etc., the body of Human β-NGF gene The analysis of outer amplification, clone and its sequence, Suzhou Medical College journal, 1999,19 (12):1271-1273.).In eukaryotic table There are more advantages up to relative to procaryotic cell expression:First, it can be processed the protein of expression, folding such as steric configuration, Glycosylation and phosphorylation etc.；2nd, the introne in foreign gene is can recognize that and removes, shearing is ripe mRNA；3rd, turn The eukaryotic of dye target gene can freeze, and stablizing expression activity destination protein after recovery, (grain scheme etc., pcDNA3-hNGF is true The structure of nuclear expression carrier and its expression in Bone Marrow Stromal Stem Cells, Chinese osteoporosis magazine, 2016,22 (1), 41-44)。

Foreign countries are expressed on a large scale with eukaryotic system, purifys NGF has a maturation process, and with prokaryotic system (E.coli) expression RhNGF2 β it is more make biological activity 50 times lower than the NGF in mouse source because cannot correctly fold, so more suggestion eukaryotic expressions System overcomes protein misfolding problem.In recent years, researcher helps its body by building the NGF with targeting sequencing Outer renaturation, although renaturation yield increases, product need to could obtain maturation NGF after limiting digestion, and increase subsequent treatment is difficult Degree (structure of Li Ping Nan etc., pIRES2-NGF-NT-3 carrier for expression of eukaryon and identification, life science, 2014,18 (4), 315-322).It is believed that comprehensive two kinds of expression systems, mammalian expression systems have the correct assembling of secretion, fold and sugar The features such as base, be more suitable for expression of NGF.

(the research of expression and its biological activity of the people B-NGF genes such as Han Xiaofeng in COS-7 cells is thin by Han Xiaofeng Born of the same parents and molecular immunology magazine, 2001,4:Transient expression, expression production 310-313.) have been carried out to NGF in COS-7 cells Thing has bioactivity, but can not realize the stabilization of NGF, long-term expression, generally only as transient expression host cell.Cause This, in genetic engineering pharmaceutical, often selects Chinese hamster ovary celI as host cell.Be conducive to using expressing cho cell foreign gene The cellular environment of disulfide formation.The expression system possesses complete transcription post-processing process, including glycosylation, carboxylated, makes The external source eukaryotic gene product of expression can keep its natural structure and activity, and mammalian cell can make its expression production Thing is secreted into extracellular culture medium, and be conducive to foreign protein isolates and purifies (Chen Yong, the structure of humanβ-NGF's carrier for expression of eukaryon Build and its bioactivity of expression product, Products in China magazine, 2005,6 (18), 457-461.).Patent 201210206171.0 recombinate nerve cell growth factor based on people prepared by expressing cho cell system is more than 5 than living after purification ×10⁵U/mg。

Therefore many research institutions all use expressing cho cell system expression recombined human NGF, determination of activity knot both at home and abroad Fruit shows, compared with mouse NGF, recombined human NGF has good bioactivity.But the biological activity of expression product and Expression is the key factor and difficult point for restricting Recombinant Naja naja atra nerve growth factor industrialization development, and the expression of the Chinese hamster ovary celI of document report It is relatively low, it is more difficult to screen overexpression cell line, highest just reaches 10mg/L or so, it is difficult to realize industrialization development (Counts SE,Mufson EJ.The role of nerve growth factor receptors in cholinergic basal forebrain degeneration in prodromal Alzheimer disease.J NeuropatholExp Neurol；2005,64(4),263-272).

In order to screen the carrier of high efficient expression, someone uses technique for gene engineering, constructs the three of growth factor of human nerve Kind eukaryotic expression plasmid, respectively pMD902- β-NGF, pcDNA3.1-His- β-NGF, pcDNA3.1-4.0-His- β- NGF removes transfection CHO cell, screening expression growth factor of human nerve, and pcDNA3.1-4.0-His- β-NGF expression quantity is most after testing Height about 28.3ng/ml (Qi Jing, different eukaryotic vectors expression growth factor of human nerve, Nanjing Medical University, 2009).There is research Use and suspended the Chinese hamster ovary celI cultivated as transfection carrier by serum-free domestication, its cell line expression quantity is reachable after screening 25.5mg/L, (find pleasure in Liu He big etc., the Chinese hamster ovary celI strain of efficiently expressing recombinant human nerve growth factor and its construction method, China Patent, 102586190 A of CN) also has the expression vector establishment mode of dual-gene coamplification of having used, make two gene magnifications The effect of collaboration is reached between system, so that NGF target gene copy numbers increase, NGF albumen obtains higher yield, warp After semisolid culturemedium cloning screens and expands culture, optimal monoclonal cell strain recombined human NGF expressions in harvest Reach 50mg/L (happy big etc., the high efficient expression of recombinant human nerve growth factor and biological evaluation research, Chinese Pharmaceutical Affairs, 2014,28 (6), 601-606).

More preferable growth factor of human nerve expression system is found, obtains higher yield, higher purity is always everybody grinds Study carefully direction.

The content of the invention

It is an object of the present invention to provide the Lentiviral of expression recombinant human nerve Porcine HGF；

It is a further object to provide the eukaryotic cell expression system containing above-mentioned Lentiviral；

It is also another object of the present invention to provide a kind of preparation method of recombinant human nerve growth factor.

By the gene insertion Lentiviral-pLX-puromycin structure weights of the recombinant human nerve growth factor Group expression vector, the secreting, expressing of recombinant human nerve growth factor is carried out using Chinese hamster ovary celI culture.

Specifically comprise the following steps：

Recombinant human nerve growth factor gene is operably connected to pLX-puromycin slow virus carriers, structure weight by I Group expression vector (as shown in Figure 3)；

The recombinant expression carrier of step I structure is carried out viral packaging by II；

Virus transfection host cell described in step II, culture are collected virion by III；

Virion transfection expression cell described in step III, acquisition can be expressed the restructuring of recombinant human nerve growth factor by IV Cell；

V collects the crude extract of the recombinant human nerve growth factor of recombinant cell expression；

The crude extract of the recombinant human nerve growth factor of VI pair of step V carries out Hydrophobic interaction chromatography, metal-chelating successively Column chromatography, ion-exchange chromatography, finally obtain the recombined human growth factor of human nerve that purity reaches 99%

Beneficial effect：The expression rate of recombinant human nerve growth factor prepared by this method reaches 30.4%；Cell supernatant In albumen reach 0.17mg/ml.In addition the sample for the mouse submandibular gland for passing through and listing, Italy (list in European Union in the recent period Product) stoste be compared discovery, the purity of rhNGF expressed and purified with this patent can reach more than 99%, than work Reach 2.5*10⁶IU/mg, higher than control sample and document report result.

Brief description of the drawings

Fig. 1 .Beta-hNGF and histidine-tagged amplification gene electrophoresis；

The endonuclease bamhi electrophoresis of Fig. 2 carriers；

The recombinant vector collection of illustrative plates that Fig. 3 structures are completed；

Fig. 4 .WB detection His-hNGF expression；

Fig. 5 .PCR verification positive colony His-hNGF expression；

The verification positive colony His-hNGF expression of Fig. 6 electrophoresis；

Fig. 7 .HIC collection of illustrative plates；

Fig. 8 .AC collection of illustrative plates；

Fig. 9 .IEC collection of illustrative plates；

Figure 10 .rhNGF protein product purity detectings；

Figure 11 .MTT methods detection protein product activity.

Embodiment

The advantages and features of the present invention is further described by the following examples, should not be understood as to the present invention's Limitation, used experimental facilities and material are common commercially available unless otherwise specified.

【Embodiment 1】The selection of hNGF genes and vector construction

Gene Name：Beta-hNGF, Genbank are numbered：NM_002506.As shown in SEQ NO.1, it is compiled its DNA sequence dna The amino acid sequence of code is as shown in SEQ NO.2.

Using slow virus carrier as template, Beta-hNGF genes are expanded with conventional PCR method, will by molecular cloning method Target gene is inserted into slow virus carrier, construction of expression vector (while being inserted into label), and 6 × His is carried so as to express N-terminal The destination protein of label, easy to purify.

1.1 gene magnification

Slow virus carrier title：PLX-puromycin (is purchased from Thermo, article No. OHS4735), label：HA-His, insertion Fragment：NM_002506；

Table 1：Design of primers

Table 2：PCR reaction systems:

Table 3：PCR response procedures

PCR carries out electrophoresis detection PCR product after reaction, with 0.8% Ago-Gel, as shown in Figure 1.Expand To hNGF Product Sequences as shown in SEQ NO.3 and His Product Sequences as shown in SEQ NO.4.

1.2. to the digestion of carrier

Table 4：Digestion system

37 DEG C of digestions 30 minutes, agarose gel electrophoresis separation and recovery, electrophoresis result are shown in Fig. 2.

1.3. the collection and restructuring of endonuclease bamhi are carried out to Ago-Gel

(1) under long-wave ultra violet lamp, the DNA bands of required recycling are cut with clean blade, excision as far as possible is free of DNA's Gel, it is the smaller the better to obtain gel volume.

(2) it will cut and be put into 1.5ml centrifuge tubes (should weigh in principle, can also estimate) containing DNA band gels, add Enter 3 times of volume sol solutions DD (about 400 μ l).

10min (or until glue is completely dissolved) is placed in (3) 56 DEG C of water-baths.Every 2~3min overturns mixing and once accelerates dissolving.

(4) previous step resulting solution being added in adsorption column EC (adsorption column is put into collecting pipe), room temperature places 1min, 12000rpm centrifuges 60sec, outwells the waste liquid in collecting pipe.

(5) 500 μ l rinsing liquids WB, 12000rpm centrifugation 30sec are added, discard waste liquid.

(6) adsorption column EC being put back in collecting pipe, 12000rpm centrifugation 2min, remove rinsing liquid as far as possible, in order to avoid rinsing liquid Middle residual ethanol suppresses downstream reaction.

(7) room temperature places 5min.

(8) adsorption column EC is taken out, is put into a clean centrifuge tube, it is ultrapure to add 30 μ l in the middle part of adsorbed film Water (heating effect is more preferable in 65~70 DEG C of water-baths in advance for ultra-pure water), room temperature place 2min, 12000rpm centrifugations 1min.

(9) fragment recombinates：Above-mentioned fragment is completed into plasmid construction by recombinating to connect, 37 DEG C recombinate 30 minutes, obtain weight Group product；

Table 5：Restructuring system

1.4. the conversion Escherichia coli TOP10 of recombinant products is used

1. using the Trans1-T1Phage Resistant Competent cells of Beijing Quan Shi King Companies, according to explanation Book carries out.

2. connection product is added in the competent cell after thawing；

3. ice bath 30min；

4. 42 DEG C of heat shock 30sec, the complete ice bath 2min again of heat shock；

5. rejuvenation：The LB culture mediums of 500ul antibiotic-frees are taken to be added thereto, 37 DEG C of constant-temperature table rejuvenation 45min；

6. apply tablet：Supernatant is removed after centrifugation, stays a small amount of culture medium to be uniformly applied to spreading rod containing anti-after cell is resuspended Property

7. on LB solid plates.37 DEG C of overnight incubations.

8. if blue hickie screening, then need to precoat X-Gal and IPTG on solid medium tablet, both according to 10:1 ratio mixes.

9. picking positive colony.

1.5. the plasmid in positive amplification clone is extracted

1. taking the bacterium solution that 1~2ml is overnight, 12000g centrifugation 2min, abandon supernatant.

2. plus 250 μ l Buffer P1, vibration mix bacterial precipitation, not remaining tiny fungus block, (Buffer P1 are in addition preceding true Recognize and added RNase).

3. plus 250 μ l Buffer P2, mixing 4~6 times of gently turning upside down, thalline is fully cracked, (this step is unsuitable More than 5min).

4. plus 350 μ l Buffer P3, mixing 6~8 times of gently turning upside down, 12000g centrifuge 10min.

5. pipe will be prepared to be placed in 2ml EP pipes (waste collection pipe), supernatant in aspiration step 4 to preparing in pipe, 12000g centrifuges 1min, abandons waste liquid.

6. will prepare pipe puts back into EP pipes, add 700 μ l Buffer PW, 12000g centrifugation 1min, abandon waste liquid.

Repeat step.

8. will prepare pipe puts back into EP pipes, 12000g centrifugations 2min.

9. being put into pipe is prepared in new 1.5ml EP pipes, natural air drying (about 5min), periosteum center plus 30 μ L are being prepared Ultra-pure water, is stored at room temperature 2~3min, 12000g centrifugations 1min.

The Vector map that structure is completed is shown in Fig. 3, and through sequence verification, sequencing result is displayed without being mutated.

【Embodiment 2】The packaging of recombined lentivirus vector

2.1.293T cell (being purchased from ATCC, CRL3216) freezes, recovers and cultivates

2.1.1 the freezing of 293T cells (nutrient solution of 293T cells is dual anti-for DMEM+10%FBS+1%) is with passage Number increase, 293T cells occur growth conditions decline, occur being mutated.So just to be carried out when cell is bought a large amount of Freeze, to ensure the stability and continuation of experiment.

1. being frozen in cell log growth period, increase cell recovery survival rate.

2. going cell supernatant, add PBS buffer and wash away remaining culture medium.

3. adding 0.05% pancreatin 1ml, digest 1-2 minutes.

4. Microscopic observation cell rounding, when intercellular gap increases, adds fresh culture 3ml piping and druming and mixes.

5. cell is centrifuged, 1000rpm, 2min.

6. adding cells frozen storing liquid (70% complete medium+20%FBS+10%DMSO) is resuspended cell, adjustment density is about For 3 × 10⁶A/ml.

7. dispensing into cell cryopreservation tube, it is put into foam box, is put into -80 DEG C of ultra low temperature freezers.

8. cell being put into liquid nitrogen and is filled for second day, and record.

9. recovery detection cell survival rate, cell state etc., and record.

2.1.2.293T the passage of cell

1) when cell growth needs to carry out passage operation to cell to converging rate and reach 80~90%, to expand cell number Amount, maintains the good growth conditions of cell.

2) 0.05% pancreatin 1ml, vitellophag 1-2 minutes are added.

3) after cell centrifugation supernatant discarding, add complete medium and cell is resuspended.

4) assign in 10cm culture dishes, 10ml/ wares.

5) 37 DEG C are put back to, is cultivated in the incubator of 5%CO2 and 95% relative humidity.

2.1.3 the recovery of 293T cells

あ is excessive (more than 30 generations) when cell passage number, when cell state is deteriorated or when contamination accident occurs in cell, needs Abandon and the cell to starting to freeze is recovered.

ぃ opens water-bath, and set temperature is 37 DEG C.

い check cell bank record, according to record from liquid nitrogen filling in take out freeze cell (mitten need to be put on, prevent by Frostbite), lose rapidly in water-bath and quickly rock, be completely dissolved cell solution in 1~2min.

1ml cell solutions are added in 9ml complete mediums and are transferred to 10cm culture dishes after mixing by ぅ.

う puts back to 37 DEG C, 5%CO₂Cultivated with the incubator of 95% relative humidity.

Second day observation cell survival rate of ぇ.Old culture medium is outwelled, adds 10ml fresh cultures.

2.2. the packaging of slow virus

Calcium phosphate transfection solution prepares：2M CaCl2 solution, 2XHBS solution (50mM HEPES, 280mM NaCl, 10mM KCl, pH 7.05)

Step

1. prepare 10cm ware 293T cells, culture medium DMEM+10%FBS, 1% Pen .- Strep in advance.

2. cell is assigned in new 10cm wares, the cell density per ware about 3E6-4E6/.

3. second day, cell is checked under mirror.Cell fusion degree should substantially 40-50%, be evenly distributed.

4. before transfection 2 it is small when, take out ware, remove original cell culture medium, add the fresh DMEM complete mediums of 8ml, will Cell sends incubator back to.

5. by taking 1 10cm ware of packaging as an example, (material is purchased from Biovector Science for prep solution A and solution B Lab)：

Solution A：Recombined lentivirus vector 10 μ g, 1 (PAX2 of packaging plasmid：10ug), 2 (VSVG of packaging plasmid：5ug), 2M CaCl₂50 μ l, with pure water constant volume to 400 μ l；(note：The order being actually added into pipe is：Water, plasmid, and CaCl₂) mix.

Solution B：400 μ l 2 × HBS solution；

6. solution A is added dropwise in solution B, mix；It is stored at room temperature 15 minutes.

7. above-mentioned A and B mixed liquors 800ul is added dropwise in the 293T cells of step 4., gently mix, place 37 degree CO₂Incubator culture 5-6h.

8. taking out the ware after transfection, supernatant discarding liquid is exhausted, replacement is preheating to 37 DEG C of complete culture solution, continues to cultivate Overnight.

9. changing 24h after liquid to collect supernatant for the first time, enter 50ml centrifuge tubes, and 10ml fresh cultureds are added into former ware Liquid, continues to cultivate.

10. regathering a supernatant after 24h, the supernatant that can be collected with first time merges.

Supernatant 3000g is centrifuged 10 minutes, collects supernatant fraction, crosses 0.45 micron membrane filter.

【Embodiment 3】The transfection and screening of recombinant slow virus particle

Slow virus infects Chinese hamster ovary celI (being purchased from ATCC, CRL-9096)

1) cell count：Chinese hamster ovary celI is according to six orifice plates, per hole 2 × 10⁵Quantity counts, overnight incubation in incubator.

2) infected cell：By the suspension of packaged lentiviral particle, (the fresh trainings of 1ml viruses+1ml are added in six orifice plates Support base).Add auxiliary reagent Polybrene to final concentration of 8 μM.After infecting overnight, next day is changed to fresh culture.

3) screen：Cell that virus infection is crossed add puromycin (Puromycin) to final concentration of 3 μ g/ml, 48 it is small when The cell not infected afterwards can mortality.

4) monoclonal and amplification cultivation are divided successful cell is infected.

5) cell extraction total protein, Western Blot, PCR verifications are collected after cell amplification.

【Embodiment 4】The His-hNGF expression of Western Blot verification cell lines

Needed according to experiment, we are using mini glue and the gradient colloid system of Amersham.

Table 6：The formula of Amersham (GE) gradient glue：

Idiographic flow is as follows：

A. board-washing, assembling：Glass plate is tried one's best wash clean, it is to be dried after assembling.

B. separation gel pours into：Mini glue is poured into glass plate after the separation gel configured directly has been added TEMED, finally Flattened with absolute ethyl alcohol；The preparation of gradient glue is respectively to take 15ml to import gradient mix device 4% and 18% separation gel, coordinates magnetic force Agitating device is poured between glass plate, is eventually formed the gradient scope of 4-18%, has been filled and flattened with absolute ethyl alcohol.

C. pouring into for glue is concentrated：It will concentrate after glue has added TEMED and add between glass plate, and choose comb as required and be inserted into Wherein, bubble is avoided.Polymerized at room temperature 20-30min.

D. loading：Ready 100 DEG C of heating 5min of protein sample, 13000rpm centrifugations afterwards 2 minutes.With albumen loading Special gun head loading, per hole 20-60ul samples.

E. electrophoresis：1 × TGS buffer are added, electrophoresis direction is from anode to cathode.

F. protein immunoblot hybridization (Western Blot, WB)

G.SDS-PAGE electrophoresis.

H. transferring film：Treat that bromophenol blue indicator is run to gel bottom and terminate electrophoresis, start transferring film.Configure containing final concentration of 20% methanol 1 × TG transferring film buffer solutions, pvdf membrane is in advance with the penetrating activation of methanol to transparence.According to filter paper-gel-PVDF The order of film-filter paper is fitted into transferring film folder, pays attention to each layer bubble as far as possible to drive away clean.Transferring film direction be from anode to cathode, Condition is constant current 400mA, and 4 DEG C chromatograph transferring film 4.5-5h in cabinets.

I. close：Transferring film finishes taking-up, and cutting corresponding band according to the size position of destination protein is put into hybridizing box.Add Enter 2% skim milk or 5%BSA closings 1h.The band of phospho-AB is closed with 5%BSA.

J. primary antibody：Add primary antibody (anti-His antibody according to certain ratio milk：1：5000, Mouse, Abmart).4 DEG C of chromatography cabinets are incubated overnight.

K. film is washed：Next day, takes out film and is put into hybridizing box.Film is washed with 0.05% PBST 3 times, and frequency is 5min/ times.

L. secondary antibody：According to the attribute of primary antibody, secondary antibody (coupling horseradish peroxidase) is added according to certain ratio, room temperature is incubated Educate 1h.

M. film is washed：Secondary antibody washes film 3 times after time is up with 0.05% PBST, and frequency is 5min/ times.

N. develop：Add Super Signal^TMWest Femto Substrate (ECL), are put into LAS-4000 and detect Chemiluminescence signal.

O.WB testing results are shown in Fig. 4.

【Embodiment 5】PCR verification positive colony His-hNGF expression and the expression rate of electrophoresis detection albumen

5.1. detection primer designs.

Design of primers principle：Sense primer is on His genes, and anti-sense primer is on hNGF.

Primer sequence：

a.Det.S：TGCTGGTTATTGTGCTGTC

b.Det.A：TTGCTCTCTGAGTGTGGTT

5.2. cell RNA extracts

WB detection positive colonies are selected, amplifying cells extraction cell total rna, comprises the following steps that：

1. removing culture medium in 6 orifice plates, PBS is washed once, adds 1ml TRIzol per hole^TMReagent cell lysis, repeatedly Piping and druming makes cell crack completely, and lysate then is moved into 1.5ml centrifuge tubes, is stored at room temperature 5min.

2. often pipe adds 0.2ml chloroforms, centrifuge tube 15sec is acutely shaken with hand, is stored at room temperature 2-3min, then 12,000 × g, 4 DEG C of centrifugation 15min.

3. solution is divided into three layers after centrifugation, 0.2ml supernatants are taken into new centrifuge tube.

4. often pipe adds 0.5ml isopropanols, and gently inhales and play several lower mixings, 10min is incubated at room temperature, then 12,000 × g, 4 DEG C of centrifugation 10min.

5. supernatant is removed, the ethanol solution washing precipitation per effective 1ml 75%, the then DEG C centrifugation of 7,400 × g, 4 5min。

6. ethanol solution is removed, naturally dry precipitation 5-10min.

7. adding the water of appropriate DEPC processing, RNA precipitate is dissolved.

5.3. cDNA is synthesized

Using the RNA that embodiment 5.2 obtains as templated synthesis cDNA, synthesized as shown in table 7.

Table 7：CDNA synthetic systems

5.4.PCR amplification

Table 8：PCR amplification condition:

Table 9：PCR response procedures

The result is shown in Fig. 5, electrophoresis detection protein expression rate to see Fig. 6 for agarose gel electrophoresis identification PCR product.

【Embodiment 6】Protein purification technique

6.1. Hydrophobic interaction chromatography (HIC)

50 chromatographic columns of XK (5x12.5cm) (GE Co.) are used for two kinds of HIC media of filling and are compared experiment, are respectively： Propyl S Sepharose 6B and butyl S Sepharose 6B (GE Co.) pillars 20mmol/L Na₂HPO₄/ NaH₂PO₄, pH 7.0 is balanced, wherein containing 8% (NH₄)₂SO₄8% (NH is contained₄)₂SO₄Above-mentioned expression rhNGF cell training Supernatant collection liquid sample introduction is supported, sample completes loading, with 20mmol/L Na₂HPO₄/NaH₂PO₄, the liquid of pH 7.0 eluted, Protein peak occurs collecting rhNGF.Chromatography collection of illustrative plates is shown in attached drawing 7.

6.2.AC chromatography

6.2.1. it is balanced with the LysisBuffer of 5 times of column volumes, filler is in the buffering identical with destination protein Under system, play the role of protected protein.

6.2.2. sample is added in the Ni NTA Beads balanced, ensures that destination protein comes into full contact with Ni2+, carry The rate of recovery of high destination protein).

6.2.3. cleaned with the Wash Buffer of 10-15 times of column volume, remove the foreign protein of non-specific adsorption, Miscellaneous liquid is washed in collection.

6.2.4. using the Elution Buffer (150mM imidazoles) of 5-10 times of column volume, protein peak is collected RhNGF. chromatography collection of illustrative plates is shown in attached drawing 8.

6.2.5. successively using the Lysis Buffer of 3 times of column volumes and the deionized water balance filler of 5 times of column volumes, most Balanced again with 20% ethanol of 5 times of column volumes afterwards.Filler is stored in the 1XPBS of 20% isometric ethanol.

7.3.IEC

The protein peak collected from metal-chelating column chromatography first passes around T Sephadex G-25 columns and changes salt, is then loaded to DEAE Sepharose F.F. (5x16.5cm) column, column 20mmol/L Tris-HCl before loading, the buffer solution of pH 8.0 A bed volume is washed with equilibrium liquid again after balance loadings, then is carried out with the 20mmol/L Tris-HCl liquid of the NaCl containing 0.12M Elution, when protein peak occurs being collected rhNGF, chromatography collection of illustrative plates is shown in Fig. 9.

【Embodiment 7】The detection of rhNGF purity of protein and activity

7.1. purity detecting

With SDS-PAGE method (resolving gel concentrations：12%；Concentrate gum concentration：5%) purity of protein (Figure 10) of detection purifying, As a result it is as shown in the table.

Table 10：Purity of protein

7.2. Activity determination

With mtt assay, TF-I (being purchased from ATCC, CRL-2003) relies on strain for cell and carries out rhNGF Activity determinations, and and The product of listing is with it has been reported that the result of (pharmacopeia three) is contrasted, and animal salivary gland sample source is in Chinese medicine biology Product examines and determine institute；The NGF sample sources of the CHO expression of listing are as shown in the table in Britain NIBSC, result of the comparison.

Table 11：

SEQUENCE LISTING

<110>Shenzhen Polytechnic

<120>A kind of expression vector, system and method for expressing recombinant human nerve growth factor

<130> SZ370-17P121742

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 726

<212> DNA

<213> artificial

<220>

<223>The DNA sequence dna of Beta-hNGF

<400> 1

atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcggaacca 60

cactcagaga gcaatgtccc tgcaggacac accatccccc aagcccactg gactaaactt 120

cagcattccc ttgacactgc ccttcgcaga gcccgcagcg ccccggcagc ggcgatagct 180

gcacgcgtgg cggggcagac ccgcaacatt actgtggacc ccaggctgtt taaaaagcgg 240

cgactccgtt caccccgtgt gctgtttagc acccagcctc cccgtgaagc tgcagacact 300

caggatctgg acttcgaggt cggtggtgct gcccccttca acaggactca caggagcaag 360

cggtcatcat cccatcccat cttccacagg ggcgaattct cggtgtgtga cagtgtcagc 420

gtgtgggttg gggataagac caccgccaca gacatcaagg gcaaggaggt gatggtgttg 480

ggagaggtga acattaacaa cagtgtattc aaacagtact tttttgagac caagtgccgg 540

gacccaaatc ccgttgacag cgggtgccgg ggcattgact caaagcactg gaactcatat 600

tgtaccacga ctcacacctt tgtcaaggcg ctgaccatgg atggcaagca ggctgcctgg 660

cggtttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720

gcctga 726

<210> 2

<211> 241

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of Beta-hNGF

<400> 2

Met Ser Met Leu Phe Tyr Thr Leu Ile Thr Ala Phe Leu Ile Gly Ile

1 5 10 15

Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile

20 25 30

Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu

35 40 45

Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala

50 55 60

Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg

65 70 75 80

Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu

85 90 95

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

100 105 110

Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe

115 120 125

His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly

130 135 140

Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu

145 150 155 160

Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu

165 170 175

Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile

180 185 190

Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val

195 200 205

Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg

210 215 220

Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg

225 230 235 240

Ala

<210> 3

<211> 747

<212> DNA

<213> Artificial

<220>

<223>The gene amplification product sequence of Beta-hNGF

<400> 3

atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcggaacca 60

cactcagaga gcaatgtccc tgcaggacac accatccccc aagcccactg gactaaactt 120

cagcattccc ttgacactgc ccttcgcaga gcccgcagcg ccccggcagc ggcgatagct 180

gcacgcgtgg cggggcagac ccgcaacatt actgtggacc ccaggctgtt taaaaagcgg 240

cgactccgtt caccccgtgt gctgtttagc acccagcctc cccgtgaagc tgcagacact 300

caggatctgg acttcgaggt cggtggtgct gcccccttca acaggactca caggagcaag 360

cggtcatcat cccatcccat cttccacagg ggcgaattct cggtgtgtga cagtgtcagc 420

gtgtgggttg gggataagac caccgccaca gacatcaagg gcaaggaggt gatggtgttg 480

ggagaggtga acattaacaa cagtgtattc aaacagtact tttttgagac caagtgccgg 540

gacccaaatc ccgttgacag cgggtgccgg ggcattgact caaagcactg gaactcatat 600

tgtaccacga ctcacacctt tgtcaaggcg ctgaccatgg atggcaagca ggctgcctgg 660

cggtttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720

gcctgaggat ccggggttgg ggttgcg 747

<210> 4

<211> 132

<212> DNA

<213> Artificial

<220>

<223>Histidine-tagged gene amplification product sequence

<400> 4

gacgatgacg ataaggaatt caccatgggg ggttctcatc atcatcatca tcatgttatg 60

gctagcatga ctggtggaca gcaaatgggt cgggatctgt acgacgatga cgataagatg 120

tccatgttgt tc 132

<210> 5

<211> 9711

<212> DNA

<213> Artificial

<220>

<223>Build the recombinant expression carrier of the Beta-hNGF completed

<400> 5

acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240

tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300

agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360

ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420

cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480

tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540

gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600

aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660

aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720

agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780

atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840

gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900

taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960

acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020

cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080

ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140

tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200

gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260

aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320

gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380

aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggttaacttt 1800

taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc 1860

aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttatcga 1920

cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 1980

tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 2040

gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 2100

ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 2160

gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 2220

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 2280

gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc 2340

cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg 2400

ggggcggggg gggggggggg gcccccccca ggcggggcgg ggcggggcga ggggcggggc 2460

ggggcgaggc ggaaaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct 2520

tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga 2580

gtcgttgcgc gctgccttcc ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc 2640

cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 2700

ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc 2760

cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg 2820

tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc 2880

gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc 2940

ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc 3000

gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc 3060

cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc 3120

gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 3180

ccgcctcggg ccggggaggg ctcgggggaa ggggcgcggc ggcccccgga gcgccggcgg 3240

ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca 3300

gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc 3360

ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg 3420

ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg 3480

gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac 3540

cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct 3600

gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgc caccatggct 3660

tacccatacg atgttcctga ctatgcgggc tatccctatg acgtcccgga ctatgcagga 3720

tcctatccat atgacgttcc agattacgct cgagatcatc atcatcatca tggtatggct 3780

agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga taagatgtcc 3840

atgttgttct acactctgat cacagctttt ctgatcggca tacaggcgga accacactca 3900

gagagcaatg tccctgcagg acacaccatc ccccaagccc actggactaa acttcagcat 3960

tcccttgaca ctgcccttcg cagagcccgc agcgccccgg cagcggcgat agctgcacgc 4020

gtggcggggc agacccgcaa cattactgtg gaccccaggc tgtttaaaaa gcggcgactc 4080

cgttcacccc gtgtgctgtt tagcacccag cctccccgtg aagctgcaga cactcaggat 4140

ctggacttcg aggtcggtgg tgctgccccc ttcaacagga ctcacaggag caagcggtca 4200

tcatcccatc ccatcttcca caggggcgaa ttctcggtgt gtgacagtgt cagcgtgtgg 4260

gttggggata agaccaccgc cacagacatc aagggcaagg aggtgatggt gttgggagag 4320

gtgaacatta acaacagtgt attcaaacag tacttttttg agaccaagtg ccgggaccca 4380

aatcccgttg acagcgggtg ccggggcatt gactcaaagc actggaactc atattgtacc 4440

acgactcaca cctttgtcaa ggcgctgacc atggatggca agcaggctgc ctggcggttt 4500

atccggatag atacggcctg tgtgtgtgtg ctcagcagga aggctgtgag aagagcctga 4560

ctcgagaatt cgaatttaaa tcggatccgc ggccgcgccc ctctccctcc ccccccccta 4620

acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt 4680

ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga 4740

cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg 4800

tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt 4860

gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat 4920

aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg 4980

aaagagtcaa atggctcacc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg 5040

taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt 5100

cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 5160

acgatgataa tatggtgagc aagggcgagg aggataacat ggccatgacc gagtacaagc 5220

ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg 5280

cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg 5340

tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg 5400

tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg 5460

cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc 5520

agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg 5580

ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc 5640

ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc 5700

gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg 5760

aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaccatca acctctggat 5820

tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 5880

ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 5940

tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 6000

caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 6060

accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 6120

ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 6180

tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc 6240

tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 6300

ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 6360

acgagtcgga tctccctttg ggccgcctcc ccgcctggta cctttaagac caatgactta 6420

caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 6480

tcactcccaa cgaaaataag atctgctttt tgcttgtact gggtctctct ggttagacca 6540

gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 6600

cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 6660

atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 6720

tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 6780

tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 6840

atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 6900

ctggctctag ctatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 6960

tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag 7020

aagtagtgag gaggcttttt tggaggccta gacttttgca gagacggccc aaattcgtaa 7080

tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 7140

cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 7200

attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 7260

tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 7320

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 7380

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 7440

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 7500

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 7560

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 7620

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 7680

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 7740

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 7800

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 7860

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 7920

actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 7980

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 8040

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 8100

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 8160

aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 8220

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 8280

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 8340

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 8400

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 8460

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 8520

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 8580

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 8640

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 8700

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 8760

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 8820

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 8880

ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 8940

tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 9000

tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 9060

gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 9120

caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 9180

atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 9240

gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 9300

tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 9360

acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 9420

gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg 9480

agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 9540

aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 9600

tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 9660

ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct g 9711

Claims (9)

1. a kind of expression vector for expressing recombinant human nerve growth factor, it is characterised in that the expression vector includes：Such as SEQ Recombinant human nerve growth factor gene described in ID NO.1, and

Lentiviral-pLX-puromycin.

2. expression vector according to claim 1, it is characterised in that the expression vector further include expression N-terminal with 6 × The HA-His labels of His label destination proteins, the label are used to purify.

3. expression vector according to claim 1, it is characterised in that the structure of the expression vector is as shown in Figure 3.

4. a kind of eukaryotic cell expression system for expressing recombinant human nerve growth factor, it is characterised in that contain claim 1 institute The expression vector and Chinese hamster ovary celI stated.

5. a kind of preparation method of recombinant human nerve growth factor, including：

1) recombinant human nerve growth factor gene is operably connected to pLX-puromycin slow virus carriers, structure is as weighed Profit requires the recombinant expression carrier described in 1；

2) recombinant expression carrier of step 1) structure is subjected to viral packaging；

3) virion is collected into the virus transfection host cell described in step 2), culture；

4) by step 3) the viral particle transduction Chinese hamster ovary celI, the eukaryotic table of structure expression recombinant human nerve growth factor Up to system；

5) incubation step 4) the recombinant cell and collect the crude extract of the recombinant human nerve growth factor of expression；

6) Hydrophobic interaction chromatography, metal chelating column layer are carried out successively to the crude extract of the recombinant human nerve growth factor of step 5) Analysis, ion-exchange chromatography, finally obtain recombined human growth factor of human nerve.

6. preparation method according to claim 5, it is characterised in that the host cell in the step 3) is thin for 293T Born of the same parents.

7. preparation method according to claim 5, it is characterised in that the Hydrophobic interaction chromatography in the step 6), is adopted Medium is Butyl-S-Sepharose FF, the preparation of the eluent of use：20mmol/L Na₂HPO₄/NaH₂PO₄, pH 7.0, wherein adding the (NH of mass fraction 8%₄)₂SO₄。

8. preparation method according to claim 5, it is characterised in that the Hydrophobic interaction chromatography in the step 6), is adopted Medium is DEAE-Sepharose FF, the preparation of the eluent of use：20mmol/L Tris-HCl, pH 8.0, wherein Add 0.12M NaCl.

9. preparation method according to claim 5, it is characterised in that the purity of the recombined human growth factor of human nerve More than 99%, reach 2.5*10 than work⁶IU/mg。