CN108300736A - The Chinese hamster ovary celI strain of efficiently expressing recombinant human β-NGF-Fc fusion proteins and its construction method - Google Patents

Abstract

The present invention relates to a kind of Chinese hamster ovary celI strain of efficiently expressing recombinant human β NGF Fc fusion proteins and its construction methods, specifically, a kind of recombinant vector and recombinant cell containing nerve growth factor Fc fusion proteins are provided, the recombinant vector, the nucleotide sequence containing nerve growth factor Fc antigen-4 fusion protein genes：PCHO1.0 beta NGF Fc such as SEQ ID NO：Shown in 1 or pCHO1.0 beta NGF opt Fc such as SEQ ID NO：Shown in 2 or pCHO1.0 beta NGF RRA Fc such as SEQ ID NO：Shown in 3, the carrier is Freedom^TMpCHO1.0.The present invention further demonstrates the growth factor of human nerve knot and Fc receptors using mammalian cell expression system successful expression growth factor of human nerve Fc fusion proteins, and sequence is correct and has higher specific activity, has a good application prospect.

Description

The Chinese hamster ovary celI strain of efficiently expressing recombinant human β-NGF-Fc fusion proteins and its structure Method

Technical field

The present invention relates to the expression vectors and cell for expressing growth factor Fc fusion proteins, and specifically, the present invention relates to And recombinant vector and recombinant cell containing growth factor of human nerve Fc fusion proteins, the invention further relates to the recombinant cells Preparation method, the purification process of recombined human β-NGF-Fc fusion proteins and the recombinant vector and recombinant cell are used to prepare recombination The purposes of humanβ-NGF's-Fc fusion proteins.

Background technology

Nerve growth factor (never growth factor, NGF) be Levi-Montalcini in 1953 in small rat meat A kind of neurotrophic factor found in oncocyte.Then, Cohen has isolated and purified out this from snake venom and mouse lower jaw gland Kind trophic factors, the neurotrophic effect shown according to it are named as nerve growth factor.Nerve growth factor It was found that having pushed the development of entire Neuscience, cell biology and Developmental Biology, the research for neural subject has been opened up newly Field.For this purpose, the prize of Nobel's medical physiology was also awarded in Levi-Montalcini in 1986.Nerve growth factor is Research is also a kind of neurotrophic factor the most thorough earliest so far.It adjust neure growth, development, differentiation, It determines all to play the role of in the direction of axon growth, the reparation of injured nerve very important.Some researches show that NGF is to dimension The function of holding the sympathetic neuron of adult is also of great significance.The disease of some retrograde neuronals, for example, Ah The occurrence and development of Zi Haimo diseases, Huntington's disease etc. are also related with the shortage of NGF.In addition to nervous system, NGF is also deposited extensively It is in Non nervous system, for example the proliferation of chromaffin cell can be promoted, the Anti-G value of bone-marrow-derived lymphocyte has research recently It was found that NGF also has positive effect in terms of the proliferation for inhibiting tumour cell.

People's ngf gene be located at No. 12 area 2 of the short arm of a chromosome with and No. 1 2nd area of the short arm of a chromosome No. 1 subzone of 1 band on, by 6 Exon and 5 introne compositions.Complete NGF be by 2 α subunits, 2 β subunits and 2 γ subunits and zinc fingers with The condensate that non-covalent bond is formed.Wherein alpha subunit belongs to non-homogeneity acid albumin, and pI is about 4.3, its effect is mainly Hydrolysis of the γ subunits to β subunits is prevented, to play a protective role to it.γ subunits have arginine ester peptide enzyme activity Property, main function is related with the synthesis of NGF, can activate inactive β subunits.β subunits are the active groups of NGF, it By two peptide chains containing 118 amino acid the dimer formed is built by non-covalent.Contain 6 half Guangs in ripe NGF chains Histidine residue can generate 3 pairs of intrachain disulfide bonds [7], and disulfide bond is particularly significant to the biological activity for maintaining NGF, once two Sulfide linkage is destroyed, NGF to be biological activity can substantially reduce and even lose.

Nerve growth factor (Never Growth Facter), is the neurotrophic factor family member being found earliest One of, there is the critical function for promoting neuron differentiation and survival, be clinically widely used in peripheral nerve injury, cornea A variety of diseases such as ulcer.The nerve growth factor listed at present is mainly derived from the lower jaw gland of mouse, and there are certain mouse source diseases Malicious pollution risk.Recombination and expression techniques are applied in this research, and people's nerve growth factor is expressed using Chinese hamster ovary (CHO) cell strain Son and IgG1Fc fusion proteins not only can avoid mouse virus pollution, and can simplify the purifying of nerve growth factor Journey, moreover it is possible to enhance the stability of recombinant protein, and effectively improve the half-life period of nerve growth factor in vivo.NGF is in vivo It is constituted in the form of 241 amino acid, normally referred to as preproNGF precursors, contains signal peptide, leader peptide and mature peptide three Part.Wherein 1-18 amino acid is signal peptide, and main function is related with the secretion of protein.In endoplasmic reticulum, prepro The signal peptide of NGF precursors is cut, and forms pro NGF precursors (223 amino acid).Pro NGF precursors are in endoplasmic reticulum Exist in the form of homodimer, be then transferred in golgiosome, the leader peptide of proNGF is removed.19-104 amino acid For leader peptide, containing there are two conservative region in leader peptide, and promote that NGF is correctly folded and the maturation outer secretion of NGF is related. ProNGF forms the ripe NGF with biological activity after the modification of N-terminal and C-terminal, in the cell, not woods egg White enzyme can be cut off leader peptide, proNGF made to be transformed into the sites Arg-Ser-Lys-Arg in specific recognition proNGF Ripe NGF.Studies have shown that proNGF does not have biological activity, the NGF ability of free N-terminal is only exposed after digestion It is combined with receptor-specific, to play biological activity.

The nerve growth factor listed at present is mainly derived from the lower jaw gland of mouse, and there are certain mouse source viruses to pollute wind Danger.This research apply recombination and expression techniques, using Chinese hamster ovary (CHO) cell strain expression growth factor of human nerve with IgG1Fc fusion proteins not only can avoid mouse virus pollution, and can simplify the purification process of nerve growth factor, also The stability of recombinant protein can be enhanced, and effectively improve the half-life period of nerve growth factor in vivo.

The present invention constructs three recombinant expression plasmids respectively, and respectively wild type ngf gene is melted with Fc sections of genes It closes, the ngf gene of codon optimization is merged with Fc sections of genes and the gene of 9 base sequences of NGF terminal deletions It is merged with Fc sections of genes.

Recombinant expression carrier is transfected respectively to CHO-S cells by liposome transfection, through Western Blot, ELISA And chick embryonic dorsal root ganglion is it is experimentally confirmed that three plants of transgenic cells can express size correctly and have preferable biological activity Recombined human β-NGF-Fc fusion proteins.

The pressure that two stages have been carried out using methotrexate (MTX) and puromycin is screened, it was demonstrated that the NGF through codon optimization Its expression quantity of cell strain is apparently higher than remaining two plants of cell.Monoclonal screening is carried out subsequently through limiting dilution assay, and is finally obtained Obtain the Chinese hamster ovary celI strain of strain capable of high-efficiency expression recombined human β-NGF-Fc fusion proteins.By this plant of cell expansion culture to 1L, collect All cell conditioned mediums purify destination protein using rProteinA affinity chromatographys.It is measured through BCA albuminimetries, this Purifying obtains destination protein 23.45mg altogether.It is found to purifying after obtained fusion protein has carried out reduction SDS-PAGE electrophoresis Visible two protein bands at 50kD and 60kD respectively use Western Blot and -terminal amino acid sequencing respectively Method pair two at protein band identify that the amino acid sequence for finding the protein band at 50kD is 15 amino acid sequences are completely the same before SSSHPIFHRGEFSVC, with NGF, and the amino acid sequence of the protein band at 60kD For 15 consensus amino acid sequences before EPHSESNVPAGHTIPQ, with NGF precursors proNGF, it was demonstrated that at this band be without The proNGF-Fc fusion proteins of Furin protease digestions.Then we using Furin protease in vitro to fusion protein again Secondary digestion confirms that proNGF precursors are cut off through Western Blot results, has obtained single purpose band.

The calibrating of obtained β-NGF-Fc fusion proteins progress physics and chemistry and biological activity will be purified：Non-reduced SDS- The purity of PAGE electrophoretic determination fusion proteins is 93.39%；Reduction SDS-PAGE electrophoresis measures fusion protein molecule 47.65kD； Fusion protein can be combined with mouse anti-human NGF monoclonal's antibody specificity；There is maximum suction at 280nm through UV scanning fusion protein Receive peak；It is 4.55~5.85 that isoelectric focusing electrophoresis, which measures fusion protein isoelectric point,；-terminal amino acid sequencing fusion protein N Hold 15 amino acid sequence SSSHPIFHRGEFSVC；Mass spectroscopy molecular weight results are 40917.20Da, with theoretical molecular weight 40917.49Da relative error is 0.0007%；Mass Spectrometric Identification has been carried out after digesting fusion protein using trypsase, there are The peptide fragment to match to 22 with destination protein, amino acid coverage rate are 77%；Chick embryonic dorsal root ganglion method measures Furin enzymes Cutting the ratio of rear fusion protein, to live be 1.04 × 105AU/mg, live as 6.54 without the ratio of the fusion protein of Furin digestions × 104AU/mg；The ratio that TF-1 cells/MTS methods measure Furin digestion rear fusion proteins is lived as 1.84 × 105U/mg；Without The ratio work of the fusion protein of Furin digestions be that cell strain mycoplasma be feminine gender to 1.07 × 105U/mg. after testing, it is inside and outside it is viral because It is sub negative, it can stablize and be passaged to for 45 generations and keep expression quantity constant.

Invention content

The purpose of the present invention is obtain the recombination of the nucleotide sequence containing nerve growth factor Fc antigen-4 fusion protein genes load Body constructs the CHO-S cell strains for capableing of excreting and expressing recombinant human β-NGF-Fc fusion proteins, and provides recombinant cell and β The preparation method of-NGF-Fc fusion proteins.

The object of the present invention is achieved like this：Recombinant vector, it is characterised in that：It contains nerve growth factor Fc fusions The nucleotide sequence of protein gene；The nucleic acid sequence of the nerve growth factor Fc antigen-4 fusion protein genes is： pCHO1.0- Beta-NGF-Fc (pCHO1.0- β-NGF-Fc expression vectors) such as SEQ ID NO：Shown in 1 or pCHO1.0-beta-NGF- Opt-Fc (pCHO1.0- β-NGF △ opt-Fc expression vectors) such as SEQ ID NO：Shown in 2 or pCHO1.0-beta-NGF- RRA-Fc (pCHO1.0- β-NGF △ RRA-Fc) such as SEQ ID NO：Shown in 3, the carrier is FreedomTMpCHO1.0.

Recombinant cell, it is characterised in that：It contains recombinant vector described in claim 1；The recombinant cell is secretion Express the CHO-S cell strains of recombined human β-NGF-Fc fusion proteins.CHO-S cell strains are in CD Forti CHO serum free mediums Middle routine culture.It is the cell after methotrexate and puromycin pressurize screening and culturing.

The pressurization screening is screened for the pressurization in two stages：First stage screening in methotrexate a concentration of 0~ 120nM；A concentration of 50~1000nM of methotrexate in second stage screening；

A concentration of 0~1.2 μ g/mL of puromycin in the first stage screening；Puromycin in second stage screening A concentration of 0~30 μ g/mL.

β-NGF-Fc fusion proteins, are prepared by above-mentioned recombinant vector or recombinant cell.

Above-mentioned recombinant vector or recombinant cell are used to prepare or produce the purposes of β-NGF-Fc fusion proteins.

The preparation method of above-mentioned recombinant cell, it is characterised in that：Include the following steps：

(1) recombinant vector of claim 1 is transfected into Chinese hamster ovary celI, in the cell culture containing serum free medium Static gas wave refrigerator 10~14 days (such as 12 days) in bottle, a concentration of 0~120nM of methotrexate, fast in the serum free medium A concentration of 0~1.2 μ g/mL of purine mycin；

(2) cell that step (1) culture obtains is transferred in shaking flask and shakes culture 11~15 days (such as the 13rd day), often Pass a generation within 3~4 days, culture medium is identical as step (1)；

(3) suitably increase cell density, continue to shake culture 18~25 days in the shaking flask containing serum free medium, often Pass a generation within 3~4 days, a concentration of 50~1000nM of methotrexate in the serum free medium, puromycin a concentration of 0~ 30 μ g/mL obtain the recombinant cell of any one of claim 2-6；

(4) optionally, further include that monoclonal screening is carried out, to obtain the cell strain of higher expression quantity to the cell obtained Process.

The preparation method of above-mentioned β-NGF-Fc fusion proteins, it is characterised in that：Preparation method including recombinant cell, and The step of cultivating recombinant cell and harvest cells and supernatant.

The purification process of β-NGF-Fc fusion proteins, it is characterised in that：Include the following steps：

1) recombinant cell of culture expression β-NGF-Fc fusion proteins, harvests cells and supernatant；

2) cells and supernatant is purified with ion-exchange chromatography, obtains sample after purification；

3) sample that step 2) obtains is purified with sieve chromatography, the β-NGF-Fc fusion proteins purified； Preferably, the above-mentioned recombinant cell of the recombinant cell of the expression β-NGF-Fc fusion proteins；It is further preferred that the recombination Cell is prepared according to the preparation method of the aforementioned recombinant cell.

The present invention successfully constructs the recombinant vector of recombined human β-NGF-Fc fusion proteins, successfully constructs recombined human β- The Chinese hamster ovary celI strain of NGF-Fc fusion proteins, and the recombinant human epidermal growth factor with correct sequence and greater activity, by This completes the present invention.

The present invention successfully constructs 3 recombinant plasmid pCHO1.0- β-NGF-Fc of structure, pCHO1.0- β-NGF △ opt- Fc、pCHO1.0-β-NGF△RRA-Fc.Its nucleotides sequence list refers to SEQ ID NO：1, SEQ ID NO：2, SEQ ID NO：3.

The present invention successfully constructs OptiPRO TMSFM- Plasmid DNA mixed liquors.

The present invention successfully constructs OptiPRO TMSFM-FreeStlyeTM MAX mixed liquors.

The present invention successfully constructs 3 kinds of carrier for expression of eukaryon, respectively pCHO1.0- β-NGF-Fc expression vectors； PCHO1.0- β-NGF △ opt-Fc expression vectors and pCHO1.0- β-NGF △ RRA-Fc expression vectors, the carrier are FreedomTMpCHO1.0.Construction of recombinant vector success is proved through bacterium colony PCR identifications, digestion identification and gene sequencing.

CHO-S cells are transiently transfected, are identified through WB and ELISA method, it was demonstrated that Transfected cells can correctly express recombined human β-NGF-Fc fusion proteins, measuring fusion protein through chick embryonic dorsal root ganglion method has preferable biological activity.Utilize first ammonia After pterin and puromycin carry out the pressure screening in two stages, the cell strain that can stablize expression recombination fusion protein is obtained, According to protein yield assessment result, finally selectes β-NGF △ opt-Fc cell strains and enter monoclonal screening.Rank is screened in monoclonal Section spreads 25 piece of 96 porocyte plates, it is observed that 527 plants of monoclonal cells are obtained, the screening wherein highest 10 plants of cells of expression quantity Expand culture, after being assessed again recombinant protein yield using ELISA, selected C6# plants of monoclonal cells enter follow-up real It tests.

Fusion protein 23.45mg is obtained altogether using rProteinA affinity chromatographys, and reduction SDS-PAGE electrophoresis showeds exist respectively It is as a result shown at two using rabbit-anti humanβ-NGF's Identification of the antibodies fusion protein ingredient there are two protein band at 50kD and 60kD Albumen can be combined with rabbit-anti humanβ-NGF's antibody specificity, it was demonstrated that albumen is NGF Related Components at two.Utilize the ends N- ammonia Preceding 15 amino acid that cardinal extremity PCR sequencing PCR measures band at 50kD is respectively SSSHPIFHRGEFSVC, with 15 ammonia before people NGF Base acid sequence is completely the same.Preceding 15 amino acid of protein band is respectively EPHSESNVPAGHTIPQ at 60kD, through compare with Preceding 15 amino acid of proNGF precursors is completely the same, it was demonstrated that protein band is NGF precursor proteins at this.Utilize Furin albumen After enzyme in vitro cuts fusion protein, single protein band is obtained.

The analysis result of β-NGF-Fc fusion proteins is as follows：Restoring SDS-PAGE electrophoretic determination fusion protein molecule amounts is 47.65kD；SDS-PAGE electrophoretic determination fusion protein purities are 93.39%；Have most at 280nm through UV scanning fusion protein Big absorption peak, the isoelectric point that isoelectric focusing electrophoresis method measures fusion protein are that 4.55~5.85, N- terminal amino groups end measures fusion Preceding 15 amino acid sequences of albumen are：SSSHPIFHRGEFSVC；The molecular weight of mass spectrometric determination fusion protein is 40917.49Da；Fusion protein carries out Mass Spectrometric Identification after trypsin digestion and obtains 22 albumen being consistent with theoretical peptide fragment altogether Matter segment, amino acid coverage rate are 77%；Chick embryonic dorsal root ganglion method measure digestion rear fusion protein specific activity be 1.04 × 105AU/mg, it is 6.54 × 104AU/mg to live without the ratio of the fusion protein of digestion；Through TF-1/MTT colorimetric method for determining digestions It is 1.84 × 105U/mg that the ratio of rear fusion protein, which is lived, and it is 1.07 × 105U/mg to live without the ratio of the fusion protein of digestion.

The beneficial effects of the invention are as follows：Successfully constructing can be with the CHO- of excreting and expressing recombinant human β-NGF-Fc fusion proteins S cell strains tentatively establish recombination fusion protein purifying process, and have carried out physics and chemistry and biological activity point to fusion protein Analysis, the research for recombined human β-NGF-Fc fusion proteins from now on are laid a good foundation.

Description of the drawings

The tomograph of Fig. 1 rhNGF.

The planar structure of Fig. 2 hNGF.

The signal of Fig. 3 .NGF goes to approach.

Gene assessment before Fig. 4 optimizations.

Gene is assessed after Fig. 5 optimizations.

Fig. 6 codons change comparison chart.

Fig. 1 .1 pCHO1.0 expression plasmid schematic diagrames.

Fig. 1 .2 over-lap PCR results.

The bacterium colony PCR identification of M DL15000DNA Marker 1-10 of Fig. 1 .3 recombinant plasmid pCHO1.0- β-NGF-Fc are not With the PCR product of monoclonal.

The bacterium colony PCR identification of M DL15000DNA Marker 1- of Fig. 1 .4 recombinant plasmid pCHO1.0- β-NGF △ opt-Fc The PCR product of 10 different monoclonals.

The bacterium colony PCR identification of M of Fig. 1 .5 recombinant plasmid pCHO1.0- β-NGF △ RRA-Fc：DL15000DNA Marker 1- 10：The PCR product of different monoclonals.

Fig. 1 .6 AvrII and BstZ171 double digestions are identified.

The expression of Fig. 2 .1 β-NGF and IgG-Fc are verified.

Fig. 2 .2 NGF content standard curves.

Fig. 2 .3 chick embryonic dorsal root ganglions measure fusion protein activity Figure 2.3Dorsal root ganglia Measured the activity of the fusion proteinStandard 1~5 are followed successively by：3AU/mL standard items, 1AU/mL standard items, 0.33AU/mL standard items, 0.11AU/mL standard items, 0.03AU/mL standard items；Blank control is transfection 48 hour cell supernatant after empty carrier；β-NGF-Fc 1~5 are followed successively by：It dilutes 45 times of cell conditioned mediums, dilute on 135 times of cells Clearly, dilute 405 3645 times of a times cell conditioned medium, 1215 times of cell conditioned mediums of dilution, dilution cell conditioned mediums are released；β-NGF △ RRA-Fc are successively For：50 times of cell conditioned mediums are diluted, 150 times of cell conditioned mediums of dilution, 450 times of cell conditioned mediums of dilution, 1350 times of cell conditioned mediums is diluted, is dilute Release 4050 times of cell conditioned mediums；β-NGF △ opt-Fc 1~5 are followed successively by：Dilute 70 times of cell conditioned mediums, dilution 210 times of cell conditioned mediums, Dilute 5670 times of 630 times of cell conditioned mediums, 1890 times of cell conditioned mediums of dilution, dilution cell conditioned mediums.

Fig. 2 .4 first stage pressure screens cell viability schematic diagram.

Fig. 2 .5 cell growth schematic diagrames.

Fig. 2 .6 protein yield schematic diagrames.

Fig. 2 .7 difference clonal expression amounts compare.

Fig. 3 .1 rProteinA affinity chromatography figures.

The reduction SDS-PAGE electrophoresis Figure3.2Reducing SDS-PAGE of Fig. 3 .2 affinity purification products Electrophoresis affinity purification product M are protein Marker, 1~No. 11 sample difference For No. 1, No. 5, No. 10, No. 15, No. 20, No. 25, No. 30, No. 35, No. 40, No. 43 samples

Fig. 3 .3 BCA measure albumen concentration standard curve.

The Western Blot identification Figure3.4Western Blot identification of Fig. 3 .4 purified products of the purified productM:Protein Marker；1~4：β-NGF-Fc fusion protein samples.

Protein band N-terminal sequencing result Figure 3.5N-terminal sequencing at Fig. 3 .5 50kD results of 50kD protein bands。

Fig. 3 .6 are protein band N-terminal sequencing result at 60kD, and the amino acid sequence measured is：1.Glu；2.Pro； 3.His； 4.Ser；5.Glu；6.Ser；7.Asn；8.Val；9.Pro；10.Ala；11.Gly；12.His；13.Thr； 14.Ile；15.Pro；It is completely the same through comparing preceding 15 amino acid sequences of protein band and proNGF at this, judge at 60kD Protein band is proNGF-Fc fusion proteins.

Western Blot qualification result Figure 3.7After Furin after Fig. 3 .7 Furin protease digestions protease cleavage Western Blot identification resultsM：Protein Marker；1:Before digestion Fusion protein sample；2：Furin protease digestion rear fusion proteins.

Fig. 3 .8 uv scans.

Fig. 3 .9 reduction SDS-PAGE electrophoresis measures molecular weight results.

Fig. 3 .10 SDS-PAGE measure fusion protein purity.

Fig. 3 .11 fusion protein isoelectric focusing electrophoresis results M：Marker；1~2：β-NGF-Fc fusion protein samples.

The N-terminal determined amino acid sequence of Fig. 3 .12 fusion proteins.

Fig. 3 .13 identification experiment results M：Protein Marker；1~2：β-NGF-Fc fusion protein samples；3：mNGF.

Fig. 3 .14 multi-charge mass spectrograms.

Fig. 3 .15 molecular weight determinations.

Fig. 3 .16 fusion protein mass spectrograms.

Fig. 3 .17 chick embryonic dorsal root ganglion methods measure fusion protein activity.

Fig. 3 .18 TF-1 cells/MTS colorimetric method for determining NGF activities.

Fig. 3 .19 liquid phases gradient tables and curve graph.

Fig. 4 .1 CHO-NGF-Fc cells and CHO-S cellular morphologies compare (A, B) CHO-NGF-Fc cells；(C、D) CHO- S cells.

Fig. 4 .2 difference generation cell NGF expression quantity P5~P45：Different generation CHO-NGF-Fc cell strains.

Fig. 4 .3 experimental group BHK-21 cells pictures (X100).

Fig. 4 .4 control group BHK-21 cells pictures (X100).

Fig. 4 .5 positive controls.

Fig. 4 .6 negative control groups.

Fig. 4 .7 CHO-NGF-Fc fluorescent stainings are tested.

Specific implementation mode

Embodiment of the present invention is described in detail below in conjunction with embodiment, but this field skill personnel will manage Solution, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Specific item is not specified in embodiment Part person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, being can With conventional products that are commercially available.

CHO-S routine cultures in CD Forti CHO serum free mediums, 1% resistive connection of addition roll into a ball agent, and when use adds Add the glutamine of 8mM.

Embodiment 1 builds recombined human β-NGF-Fc fusion protein carrier for expression of eukaryon.

1.1 main agents and instrument.

AvrII, BstZ17I restriction enzyme are purchased from U.S. NEB company's T 4DNA ligases；Purchased from NEB companies of the U.S. DL15000, DL6000DNA molecular weight Marker are purchased from TaKaRa companies of Japan；Q5High-Fidelity PCR kits are purchased from NEB companies of the U.S.；Wizare Plus SV Minipereps DNA Purifcation System；Purchased from Promega companies； QIAquick Gel Extraction Kit are purchased from QIAGE companies；LB liquid medium：Peptone powder 10g, yeast extraction Object：5g, sodium chloride 10g add deionized water to 1L, spare after high pressure sterilization；PCHO1.0 carrier for expression of eukaryon is purchased from Gibco Company, -20 DEG C of refrigerators preserve；E.coli DH5a competent cells are purchased from transgen companies.

1.2 experimental method.

1.2.1 the clone of growth factor of human nerve, growth factor of human nerve optimization gene and Fc sections of genes.Using full genome Synthetic method, according to the cDNA sequence (GeneBank of the GeneBank growth factor of human nerve provided:), NM-002506 people CDNA sequence (the GeneBank of Fc sections of IgG1：NM-000586.3 the DNA sequence dna of the growth factor of human nerve) and by optimization It send to Tai He Bioisystech Co., Ltd of Sino-U.S. and carries out full genome synthesis.The gene of synthesis is connected in pMD18T carriers, i.e., PMD18T-nerve gowth factor carriers and pMD18T-nerve gowth factor △ opt and pMD18T- are obtained IgG1 Fc carriers.The DNA sequence dna of synthesis is compared and analyzed with objective gene sequence using Bioedit biological softwares.

1.2.2 the primer is tested,

NGF-F 5’-TGCAGCCTAGGGCCACCATGTCCATGTTGTTC-3’

IgG1Fc-R 5’-AGGACTTGGGCTCGGCTCTTCTCACA-3’

NGF-Fc M1 5’-AGGACTTGGGCTCGGCTCTTCTCACA-3’

NGF-Fc M2 5’-TGTGAGAAGAGCCGAGCCCAAGTCCT-3’

NGF△RRA-Fc M1 5’-AGGACTTGGGCTCCACAGACTTCCTG-3’

NGF△RRA-Fc M2 5’-CAGGAAGTCTGTGGAGCCCAAGTCCT-3’

NGF△opt-F 5’-TGCAGCCTAGGGCCACCATGAGCATGCTGTTC-3’

NGF△opt–Fc M1 5’-AGGACTTGGGCTCGGCGCGGCGCACGGCCTTGC-3’

NGF△opt–Fc M2 5’-TCCTGAACCCGAGCCGCGCCGCGTGCGGAACG-3’

1.2.3 the acquisition of target gene.

1.2.3.1 the acquisition of β-hNGF-Fc fusions.,

1.2.3.1.1 expanding β-hNGF genes

After taking plasmid pMD18T-nerve gowth factor carriers to dilute 100 times, gene expansion is carried out according to following systems Increase:

Reaction condition：37 DEG C of water-bath digestions 6 hours, 1.0% Ago-Gel, 100V voltages, electrophoresis 40 minutes.

1.2.6.2 connection reaction

Reaction condition：16 DEG C of metal baths, connection is overnight

Terminate reaction：65 DEG C of heating 10min

1.2.6.3 the conversion of competent cell DH5 α

DH5 α competent cells are taken out from -70 DEG C of refrigerators, are placed 10min on ice, so that it is melted and connection product is added 50 μ L, mixing place 30min on ice, are added immediately without any with 42 DEG C of water-bath heat shock 90s then at 2min is placed on ice LB culture mediums 800 the μ L, 37 DEG C, 150rpm cultures 45min of antibiotic.5000rpm centrifuges 2min, discards 500 μ L supernatants, will Remaining bacterium solution is equably applied in the LB tablets of the kanamycins containing 100 μ g/ml, is inverted and was cultivated in 37 DEG C of incubators Night.

1.2.6.4 positive colony select and bacterium colony PCR identification

The positive bacterium colony on kanamycins LB tablets is selected with thin pipette tips, then in the liquid containing 100 μ g/ml kanamycins In body LB selective mediums, 37 DEG C are expanded, and remaining thalline is seeded in PCR system, logical using pCHO1.0 plasmids PCR identifications, the same 3.1.1 of reaction system are carried out with primer pair bacterium colony.PCR reaction conditions are：98 DEG C of pre-degeneration 30s；98 DEG C of denaturation 10s；60 DEG C of annealing 30s；72 DEG C of extension 27s；72 DEG C of extension 10min after 28 cycles.After electroresis appraisal, PCR results are chosen It is expanded for positive bacterium colony, 37 DEG C, 200rpm carries out small amount plasmid extraction after cultivating 14h.

1.2.6.5 the extraction of plasmid

The bacterium solution 1mL being incubated overnight is taken, is added in 1.5mL centrifuge tubes, 12000rpm centrifuges 1min, carefully discards upper liquid Body repeats the above steps, and often 5~10mL of bacterium solution is added in pipe

Buffer I (Cell Resuspension Solution) solution is added into the centrifuge tube there are bacterial sediment 250 μ L are blown and beaten 5~10 times with pipettor, thalline are made fully to be resuspended

250 μ L Buffer II (Cell Lysis Solution) solution are added into centrifuge tube, slowly turn upside down Centrifuge tube 5~10 times, makes the bacterium in centrifuge tube fully crack, and should see bacterium solution at this time becomes very sticky

10 μ L of Proteinase K are added into centrifuge tube, slowly turn upside down centrifuge tube 5~10 times, it is made to mix well (the above-mentioned 3.4 step used time was preferably not more than 5 minutes)

350 μ L Buffer III (Neutralization Solution) solution are added into centrifuge tube, turn upside down Centrifuge tube 10~20 times, makes solution mix well, at this time visible apparent cotton-shaped white precipitate in test tube.

Above-mentioned centrifuge tube is put into centrifuge, 10000rpm, centrifuges 10 minutes, supernatant is carefully moved into pipette In adsorption column, 10000rpm is centrifuged 2 minutes, discards solution in lower layer's centrifugation.

500 μ L of Column Wash Solution are added to adsorption column, stands 5 minutes, 10000rpm, centrifuges 2 minutes, Liquid in next centrifuge tube is discarded, is repeated the above steps 1 time.

It by adsorption column 10000rpm, dallies 2 minutes, remaining Column Wash Solution in adsorption column is made to remove

100 μ L of sterile water for injection are added into adsorption column, are stored at room temperature 5min, 10000rpm, centrifuge 1 minute, collect Plasmid solution measures plasmid concentration with micro-spectrophotometer.

1.2.7 digestion identification and sequencing

1.2.7.1 the digestion identification of plasmid

The clone of the bacterium colony PCR positives and empty carrier pCHO1.0 are subjected to double digestion identification, digestion system is as follows：

Reaction condition：37 DEG C of water-bath digestions 6 hours, 1.0% Ago-Gel, 100V voltages, electrophoresis 40 minutes.

1.2.7.2 sequencing

It is that positive plasmid is sent to the sequencing of Tai He biotech companies of Beijing Sino-U.S. by qualification result.

1.3 result

1.3.1 recombinant plasmid schematic diagram

Fig. 1 .1 are the schematic diagram of expression vector pCHO1.0, which can express a gene or using above carrier Hybridize CMV promoter and co-expresses 2 target gene.The carrier include dihyrofolate reductase (DHFR) selected marker and Puromycin (puromycin) resistant gene, can be screened using MTX and puromycin simultaneously, which also contains card That mycin resistant gene, this research by target gene be inserted into AvrII restriction enzymes and BstZ17l restriction enzymes it Between.

1.3.2 over-lap PCR result

Fig. 1 .2 over-lap PCRs are as a result, the theoretical size of people's ngf gene is 723bp, the theoretical size of-Fc sections of genes of human IgG1 For 699bp, the theoretical size of people's ngf gene segment after codon optimization is also 723bp, and people's NGF C-terminals lack 3 The theoretical size of the gene of amino acid is 714bp, so recombined human β-NGF-Fc β-NGF △ opt-Fc and β-NGF △ RRA-Fc The theoretical size of fusion segment is respectively 1422bp, 1422bp and 1413bp.PCR is obtained it can be seen from Fig. 1 .2 results It is consistent with theoretical size to purpose band between 1000bp and 2000bp.

1.3.3 bacterium colony PCR results

The bacterium colony PCR identification bacterium colony PCR identification of M DL15000DNA of Fig. 1 .3 recombinant plasmid pCHO1.0- β-NGF-Fc Marker 1-10.The bacterium colony PCR identification of M DL15000DNA of Fig. 1 .4 recombinant plasmid pCHO1.0- β-NGF △ opt-Fc Marker 1-10.The bacterium colony PCR identification of M of Fig. 1 .5 recombinant plasmid pCHO1.0- β-NGF △ RRA-Fc：DL15000DNA Marker 1-10：The PCR product of different monoclonals.The PCR product of different monoclonals.The PCR product recycling of different monoclonals PCR product carries out AvrII, BstA17I double digestion with carrier pCHO1.0, recycles digestion products, the double enzymes of T4DNA ligases connection Carrier after cutting and target gene fragment, transformed competence colibacillus E.coliDH5 α carry out bacterium after selecting the monoclonal activation after conversion PCR identifications are fallen, according to bacterium colony PCR as a result, the clone for choosing the PCR result positives carries out digestion identification after being enlarged culture.

1.3.4 the digestion identification of recombinant plasmid

Fig. 1 .6AvrII and BstZ171 double digestion qualification results.To 3 recombinant plasmid pCHO1.0- β-NGF- of structure Fc, pCHO1.0- β-NGF △ opt-Fc, pCHO1.0- β-NGF △ RRA-Fc use AvrII and BstZ171 restriction enzymes respectively Enzyme has carried out digestion identification, and digestion products have carried out 1% agarose gel electrophoresis, the result is shown in Figure 1 .3, the recombination after digestion For carrier in the visible two apparent bands in the left and right positions 1500bp and 13000bp, purpose band and theoretical size are almost the same.And 1 band is only seen after digestion without the pCHO1.0 carriers of transformation, stripe size is about 13000bp.

1.3.5 sequencing result

It takes the recombinant plasmid that bacterium colony PCR and digestion qualification result are positive to send to calm and peaceful biotech company of Beijing Sino-U.S. to survey Sequencing result is compared sequence with target gene, as a result confirms the sequencing result and theoretical sequence complete one of 3 kinds of recombinant plasmids It causes, sequence alignment figure is shown in annex.

1.4 brief summary

β-NGF-Fc, β-NGF △ opt-Fc, β-NGF △ RRA-Fc fusion gene sequences has successfully been obtained, after double digestion Success connects gene order in pCHO1.0 expression vectors, is identified through bacterium colony PCR, after digestion identification and sequencing identification, correctly It is consistent with theoretical sequence to construct 3 recombinant expression plasmids.

Structure stablizes the cell strain of expression recombined human β-NGF-Fc fusion proteins.

2.1 experiment materials and reagent.

2.1.1 main material and reagent；Serum free medium CD Forti CHO, it is public purchased from Life technologies Department；Transfection reagent FreeStlyeTM MAX Reagent are purchased from Life technologies companies；Culture medium OptiPRO TMSFM is purchased from Life technologies companies；Methotrexate (MTX), MTX are purchased from Sigma；Puromycin, Puromycin, purchase From Life technologies companies；Cells frozen storing liquid, Synth-a-Freeza CTS are purchased from gibco companies；Resistive connection group Agent, Anti-Clumping Agent are purchased from gibco companies；Glutamine, Glutamine are purchased from gibco companies；CHO-S is thin Born of the same parents are preserved purchased from Gibco companies are purchased from by this section office (National Institute for Food and Drugs Control's reconstituted drug room)；DMEM is cultivated Base is purchased from gibco companies；T75, T24 Tissue Culture Flask, tri- horn cells shaking flask of 125mL, 1L, 15mL, 50mL centrifuge tube are purchased From Corning companies；96 holes, 24 holes, 6 porocyte culture plates are purchased from Corning companies.

2.2 experimental method.

2.2.1 recombinant expression plasmid transiently transfects CHO-S cells.

2.2.1.1 the culture of cell, pass on and freeze.

The culture and passage of CHO-S.

CHO-S routine cultures in CD Forti CHO serum free mediums, 1% resistive connection of addition rolls into a ball agent, thin using 125mL Born of the same parents' shaking flask, 130~150rpm, are cultivated in 8%CO2 by 37 DEG C.Whole cell suspensions are moved in 50mL centrifuge tubes when passage, 1000rpm, centrifuge 5min, then be added CD Forti culture mediums be resuspended, the density passed on every time be 3 × 105/mL, every 3~5 It passes a generation.

CHO-S's freezes and recovers

It takes the CHO-S cells in exponential phase in 125ml cell shaking flasks, 1000rpm to centrifuge 5min, discards thin Born of the same parents' supernatant is added after 3mL cells frozen storing liquids are resuspended and cell suspension is added in cell cryopreservation tube, is first placed in mistake in -80 DEG C of refrigerators Night, transfer in second day are set in liquid nitrogen container and are preserved.When cell recovery, cell is taken out from liquid nitrogen container, it is first determined Cell Name and Cryopreservation tube is put into 37 DEG C of warm water and melts rapidly, cryopreservation tube is opened in super-clean bench by generation, is hanged cell with 1mL pipettors Mixing in the CD Forti culture mediums containing 14mL is added in liquid, and 1000rpm centrifuges 5min, discards supernatant liquid, extremely by cell inoculation It in 75cm2 Tissue Culture Flasks, is cultivated in 37 DEG C, 8%CO2 cell incubators, routine observation cell growth condition.

2.2.2 endotoxin plasmid is gone to carry greatly

Bacterium colony containing correct plasmid is incubated overnight 200mL bacterium solutions, uses QIAGEN EndoFree Plasmid Giga kit carry out plasmid extraction.

LB bacterium solutions overnight are collected, 1000rpm, centrifuges 15min by 4 DEG C, 100mL Buffer P1 is added, thalline is resuspended

100ml Buffer P2 are added, fully overturn mixing 4~6 times, are placed at room temperature for 5min, 100mL precoolings are added Buffer P3, fully reverse mixing 4~6 times (exerting oneself).

QIAfilter cartridges are connected into screw socket vial, vacuum pump is connected, lysate is poured into QIAfilter cartridges, are placed at room temperature for 10min.Liquid is set by filter membrane, to be added 50ml's completely using vacuum pump Buffer FWB2 are precipitated with sterile glass rod gentle agitation, and reusing vacuum pump makes liquid pass through filter membrane completely.

It is added in the Buffer ER to filtered solution of 20mL, overturns mixing 4~6 times, place 30min on ice.

QIAGEN-tip is balanced with 100mL Buffer QBT, is placed naturally, the tip until liquid is entered completely, then by 5 QIAGEN-tip is added in the filtered solution of step gained, is placed at room temperature for, makes it completely into tip.

QIAGEN-tip is washed using the Buffer QC of 500mL

Using the Buffer QN eluted dnas of 35mL, the isopropanol precipitating DNA, 15000rpm, 4 DEG C of 25mL are added, from After heart 30min, discard supernatant

10mL is added washs DNA, 15000rpm without endotoxic 70% ethyl alcohol, centrifuges 10min

30min is spontaneously dried, with the endotoxin-free Buffer TE dissolving DNAs of suitable volume.

2.2.3 transiently transfecting

Cell prepares before transfection：Transfection first 24 hours, using the CD Forti CHO culture mediums containing 8mM glutamine into Row cell passes on, and inoculum density is：5 × 105~5 × 106/mL, condition of culture are 37 DEG C, and 8%CO2,130~150rpm suspend Culture, before transfection two generations must remove resistive connection group agent.

The preparation of plasmid：Transfection is 1~5 μ g/ μ L of concentration with plasmid, it is necessary to clean, sterile, without organic matter solvent and chlorination The inorganic salts such as sodium pollute.

The preparation of transfection cell suspension：Cell is taken out from incubator before transfection, is obtained using the method that cell shunts Single cell suspension, sampling count, cell viability need to reach 95% or more at this time, cell suspension is adjusted to 1 with dilution method × 106/mL is put into incubator and shakes culture until transfection.

It is prepared by OptiPRO TMSFM- Plasmid DNA mixed liquors：The OptiPRO of 1.45mL is added in 50mL centrifuge tubes TMSFM adds the plasmid of 50 μ g, total volume 1.5mL, soft mixing.

The preparation of OptiPRO TMSFM-FreeStlyeTM MAX mixed liquors：Using preceding first by FreeStlyeTM MAX Reagent lightly turns upside down mixing.50mL centrifuge tubes are removed, 1.45mL OptiPRO TMSFM are added, add 50 μ L The soft mixings of FreeStlyeTM MAX Reagent.

It is with pipettor that mixed liquor is slow immediately after OptiPRO TMSFM-FreeStlyeTM MAX mixed liquors prepare The mixing of OptiPRO TMSFM- Plasmid DNA is added, slowly overturns mixing, is stored at room temperature 15min, keeps Plasmid DNA-liposome multiple Zoarium is formed.

The DNA-FreeStlyeTM MAX mixed liquors of above-mentioned 3mL are drop by drop added in cell shaking flask. while adding Shake kinetocyte shaking flask.

Cell after transfection is put back in incubator and continues culture 48 hours, condition of culture is：37 DEG C, 8%CO2,130~ 150rpm shakes culture.

2.2.4Western the expression of blot methods identification β-NGF-Fc fusion proteins

The collection of sample

Cell after transfecting 48 hours is collected, 1000rpm centrifuges 5min, and supernatant collection is placed on ice for use, Cell pyrolysis liquid RIPA 2mL are added in remaining cell, and 50 μ L of PMSF are added, and mixing places 30min, repeatedly mixing 4 on ice ~5 times, 1000rpm, centrifugation 5min removes cell fragment, and careful absorption supernatant is placed in spare on ice.Transfect empty plasmid CHO-S cells are same as above as negative control, processing method.

Restore SDS-PAGE

It takes cell supernatant and each 20 μ L of cell pyrolysis liquid in 1.5mL centrifuge tubes, 4 × Loading Buffer 5 is added 2 μ L of μ L, Reducing Agent, mixing.Boiling water heats 2min, 6000rpm, centrifuges 1min, 4~12%Bis-Tris Gel Per 25 μ L, the 200V voltage stabilizings of hole loading, electrophoresis 35min.

Transferring film

Transferring film is carried out using invitrogen iBlot albumen half dry type electroporations, is sequentially placed into mating turn from bottom to up Film bottom, gel, filter paper, transferring film top and sponge then take out pvdf membrane, just according to transferring film instrument program transferring film 7min after electrophoresis Face marks.

The incubation of antibody

Closing：Pvdf membrane is put into confining liquid (the TBST buffer solutions containing 5% skimmed milk power) and closes 1h.

Primary antibody：Use fresh confining liquid successively 1:500 dilution rabbit-anti growth factor of human nerve monoclonal antibodies, mouse anti human The former two is distinguished incubated cell supernatant samples, GAPDH mono- by IgG1Fc monoclonal antibodies, the anti-GAPDH monoclonal antibodies of mouse Anti- incubated cell lysate sample, 4 DEG C of overnight incubations.

Wash film：The TBST washing lotions containing 0.11% polysorbas20 are prepared, 10min/ times, wash film 4 times

Secondary antibody：Use fresh confining liquid successively 1:The goat anti-rabbit igg antibody of 10000 dilution HRP labels, goat anti-mouse igg Antibody is incubated at room temperature 1h.

10min/ times, prepare chromogenic reaction after washing film 4 times.

Chromogenic reaction

Color developing agent Peroxide Buffer and Enhancer Solution are pressed 1:1 mixing, drops evenly on PVDF, After being protected from light colour developing 5min, it is put into LAS-3000 photographic systems and takes a picture.

2.2.5ELISA method measures the content of NGF in supernatant

The preparation of sample and reagent：

Sample：Cell suspension is collected, 1000rpm centrifuges 10min, collects in 1mL cell conditioned mediums to 1.5mL centrifuge tubes, 13000rpm, centrifuge 10min, collect supernatant, according to 10 times of doubling dilutions to concentration in 2000pg/mL.Washing lotion：Take 20 × Concentration washing lotion 50mL is poured into bottle, and deionized water 950mL is added, and is uniformly mixed, and it is spare to be placed in 4 DEG C of refrigerators, 1 month shelf-life. NGF standard items：It is redissolved with 500 μ L dilutions, in -20 DEG C of preservations after the packing of 100 μ L/ branch, the term of validity 18 months, when use is used Dilution presses 1:500 dilutions, matching while using.

The dilution of standard items and sample-adding

16 hole of setting standard sample wells, is separately added into 100 μ L of standard items, so in the first, second hole on enzyme mark coating plate 50 μ L of standard dilutions are added in the first, second hole afterwards, blow and beat mixing；Then 100 μ L are respectively taken from the first hole, the second hole It is added separately in third hole and the 4th hole, then 50 μ L of standard dilutions is added in third hole, the 4th hole, blow and beat mixing； And so on, finally respectively take 50ul liquid to discard in 15 holes of mixing, 16 holes.(the sample-adding amount in hole is 50 after dilution The concentration of μ L, standard items are followed successively by 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/ mL、15.625pg/mL).Control wells (the CHO-S cell conditioned mediums of transfection empty carrier), sample to be tested hole are set when sample-adding.By sample It is added on the bottom in ELISA Plate hole, hole wall is not touched as possible, gently shakes mixing.

It is incubated：ELISA Plate after sample-adding is placed on 37 DEG C of incubators with sealing plate film sealing plate and is incubated 1 hour.

Board-washing：It carefully takes sealing plate film off, discards liquid, dry, 300 μ L of cleaning solution are added per hole, are discarded after static 30 seconds, It is so repeated 3 times, pats dry.

It is enzyme：50 μ L of enzyme marking reagent are added per hole, 37 DEG C of incubators are incubated 1 hour, board-washing 3 times.

Colour developing：Developing solution A50 μ L, developing solution B50ul are added per hole, gently vibrates mixing, 37 DEG C of incubators are protected from light colour developing 30 Minute.

It terminates：50 μ L of terminate liquid are added per hole, terminate reaction.

It measures：It is returned to zero with the holes Blank, 450nm wavelength measures the OD values in each hole.

2.2.6 chick embryonic dorsal root ganglion method surveys fusion protein activity

2.2.6.1 the preparation of Collagen type-I prepares 0.1% acetum, after 10 pounds of 10 minutes high pressure steam sterilizations It is spare；250 grams of weight big white mouse one are taken, rat-tail is cut off from tail portion, 75% alcohol is placed in and impregnates 30 minutes；Rat-tail is cut into 1.5 centimetres or so of segment, peels off fur, and extraction tail tendon is placed in plate；Tail tendon is shredded, the acetum of 150mL is immersed, sets 4 DEG C of refrigerators, and vibrate frequently；After 48 hours, dissolved tail tendon is moved into sterile centrifugation tube, with 4 DEG C, 4000rpm, centrifugation 30 minutes；In the every 500 μ L separating device centrifuge tubes of Aspirate supernatant, -20 DEG C of stored frozens.

2.2.6.2 activity determination method

Glass Tissue Culture Flask (each sample needs 5 Tissue Culture Flasks) after high pressure of learning from else's experience sterilization treatment, will freeze After the Collagen type-I deposited melts, 50 μ L are taken according to each cell bottle, lower the 1/3 of vial bottom surface is uniformly applied to elbow glass bar Place, removes bottle cap, is placed in super-clean bench 48 hours dry；The DMEM trainings containing 10%FBS of 2mL are added into each culture bottle Base is supported, covers bottle cap, the soaked overnight in super-clean bench；When experiment, ganglionic instrument will be dissected and be dipped in physiological saline, solved The dorsal root ganglion for taking instar chicken embryos on the 8th under mirror is cutd open, the culture medium in culture bottle is outwelled, neuromere is inoculated in and is coated with rat-tail glue In former culture bottle, 3 neuromeres of every bottle of inoculation are cultivated 2 hours in 37 DEG C of incubators；It will be active with plasma-free DMEM medium Reference material is diluted to 3AU/mL, and sample is diluted to 3ng/mL；Sample and reference material do 3 times times with plasma-free DMEM medium Than dilution, reference material and sample all do 5 dilutions (should be diluted to and negative findings occur), are added in 2mL liquid to culture bottle, And set up negative control (plasma-free DMEM medium)；37 DEG C are placed in, after culture 24 is small in 5% carbon dioxide incubator, micro- Under the microscope as a result, according to dorsal root ganglion enation in various degree do classification record (with #, ++++, +++, ++, ++ ,- Indicate result).

2.2.7 first stage pressure is screened.

2.2.7.1MTX the preparation of solution.The 1M NaOH of 100~150 μ L are added in precision weighing MTX powder 10mg, because MTX is easy inactivation at pH to higher environment, as soon as so observing that MTX powder is completely dissolved, it is 7.0 to be rapidly added pH PBS solution 22mL, vibrate mixing.It using 0.22 μM of membrane filtration degerming, takes 1.5mL centrifuge tubes several, is distributed into every pipe 100 μ L, -80 DEG C of refrigerators preserve, current existingization.

2.2.7.2 the selection of screening reagent MTX concentration

The CHO-S cells of logarithmic growth phase, 800rpm, centrifuge 5min, collect cell, using 37 DEG C preheat contain 10% resistive connection rolls into a ball agent, and cell is resuspended in the CD Forti serum free mediums of 8mM glutamine, and cell counter counts, by cell Density is adjusted to 1 × 104/mL, and the holes 1mL/ are seeded in 24 porocyte culture plates, is put into 37 DEG C, 8%CO2 cell incubators Middle culture is for 24 hours.After for 24 hours, observation cell density rises to about 3 × 105/milliliter, and MTX solution is added, concentration is made to be followed successively by 0nmol/L、20nmol/L、40nmol/L、60nmol/L、80nmol/L、100nmol/L、110nmol/L、120nmol/L.It will Tissue culture plate puts back to 37 DEG C, continues to cultivate in 8%CO2 cell incubators.Observation cell death situation every three days, every 3~4 It replaces fresh culture, keeps MTX concentration constant, and the minimal lethal dose of cell is the working concentration of MTX.

The selection of screening reagent Puromycin concentration

The CHO-S cells of logarithmic growth phase, 800rpm, centrifuge 5min, collect cell, using 37 DEG C preheat contain 10% resistive connection rolls into a ball agent, and cell is resuspended in the CD Forti serum free mediums of 8mM glutamine, and cell counter counts, by cell Density is adjusted to 1 × 104/milliliter, and the holes 1mL/ are seeded in 24 porocyte culture plates, is put into 37 DEG C, 8%CO2 cell culture It is cultivated for 24 hours in case.After for 24 hours, observation cell density rises to about 3X105/milliliter, and Puromycin solution is added, makes concentration It is followed successively by 0 μ g/mL, 2 μ g/mL, 4 μ g/mL, 6 μ g/mL, 8 μ g/mL, 10 μ g/mL, 1.1 μ g/mL, 1.2 μ g/mL.By cell culture Plate puts back to 37 DEG C, continues to cultivate in 8%CO2 cell incubators.Cell death situation is observed every three days, is replaced within every 3~4 days Fresh culture keeps Puromycin concentration constant, and the minimal lethal dose of cell is the working concentration of Puromycin.

2.2.7.4MTX, Puromycin pressurizations screening.

Cell for 24 hours after transfection, 1000rpm is taken to collect cell after centrifuging 5min and count, cell density is adjusted to 5 × 105/mL, 30mL/ bottles are seeded in T150 Tissue Culture Flasks, and MTX and Puromycin is added, makes its final concentration be respectively Cell is put back to 37 DEG C, 8%CO2, humidity static gas wave refrigerator in 80% or so incubator by 100 nmol/L, 10 μ g/mL.Often A small amount of cell suspension was taken every two days, records its cell viability.The 10th day of pressure screening in the first stage, cell viability is down to most Low, subsequent cell viability bottom out, the 14th day of pressure screening, cell viability restore to 40% or more, incite somebody to action in the first stage Cell is collected, and cell concentration is adjusted to 3 × 105/milliliter, is seeded in 125mL cell shaking flasks, maintenance MTX, The concentration of Puromycin is constant, and cell is put back in incubator, and 130~150rpm, which suspends, to be cultivated.Every 3~4 days to cell into Row passage, the cell-seeding-density passed on every time are 3 × 105/mL, maintain the concentration of MTX, Puromycin constant.First The 23rd day of staged pressure screening, cell viability restore to 85% or more, and viable count is more than 1 × 106/mL, first stage Screening is completed, and every bottle of cell cryopreservation 5 is managed, remaining cell enters the screening of second stage pressure.

2.2.8 second stage pressure is screened

The mixing clonal cell line that first stage screens is inoculated into two 125mL Tissue Culture Flasks, body is cultivated Product is 30ml, and inoculum density is 3 × 105/mL.MTX to 50nnol/mL, Puromycin to 30 μ are added in first shaking flask g/mL.MTX to 1000nnol/mL, Puromycin to 30ug/mL are added in second shaking flask.Above-mentioned shaking flask is positioned over 37 DEG C, in 8%CO2 cell incubators, 130~150rpm shakes culture, and sampling in every two days counts, and liquid is changed in centrifugation weekly, keeps sieve Select pressure constant, when the sign restored, beginning passage screening in next step is presented in cell viability.The cell every 3~4 screened in shaking flask Its passage is primary, and the cell-seeding-density passed on every time is 3 × 105/mL, when viable count is more than 6 × 105/mL, adopts With diluted passage method, appropriate MTX, Puromycin are added to maintain pressure constant according to the amount for the culture medium being newly added.Second The 39th day of staged pressure screening, cell viability are all higher than 90%, and mark second stage pressure screening terminates, each cell cryopreservation 5 pipes, remaining cell proceed by expression quantity evaluation.

2.2.9 monoclonal screens

2.2.9.1 limiting dilution assay obtains monoclonal cell

It is several to prepare 96 porocyte culture plates, 50mL centrifuge tubes, 1.5mL centrifuge tubes, marks respectively.It collects all thin Born of the same parents, 1000rpm, centrifugation 5min, using containing 8mM glutamine, 100nmol/LMTX, 10 μ g/mLPuromycin, 1% is anti- Cell is resuspended the CD Forti serum free mediums 10ml of conglomeration agent, is counted after blowing and beating mixing.It is 1.8 to measure cell density × 106/mL, cell suspension 1ml is taken to be added in 15ml centrifuge tubes, 800 μ L of culture medium are added, blows and beats mixing, obtaining density is The cell suspension of 1.0 × 106/mL.From taking 1ml to be added in 50mL centrifuge tubes in above-mentioned cell suspension, it is fresh to add 9mL Culture medium fully blows and beats mixing, obtains the cell suspension that density is 1.0 × 105/mL.It repeats the above steps three times, obtains close Degree is the cell suspension of 100/mL.It takes in above-mentioned cell suspension 2ml to 50mL centrifuge tubes, 38mL fresh cultures is added, twist Tight bottle cap, fully reverse mixing contain 1 cell to get being 5/mL to cell density per 200ul.In 96 hole cell culture In the hole of edge 36 of plate, 200 holes μ L/ of blank cultures are added per hole respectively, are added respectively per hole in intermediate 60 holes 200 holes μ L/ of cell suspension, are obtained 25 96 porocyte culture plates.After the completion of plating cells, immediately under the microscope one by one into Row observation, will have in hole and the hole of only 1 cell is marked, as monoclonal hole.Without containing cell or there are two containing And the hole of more than two cells is labeled as discarded hole, is subsequently no longer observed, 527 monoclonal cells are obtained at this time Hole.96 porocyte culture plates after observation are put back in incubator, 37 DEG C, 8%CO2 stationary cultures 20 days.96 holes are taken out after 20 days Tissue culture plate, to monoclonal hole observation period growing state one by one under the microscope, wherein 392 plants of monoclonal cell strains are able to Growth, we select the faster 240 plants of monoclonal cells of the wherein speed of growth, are seeded in 24 porocyte culture plates, often The fresh culture of 1.5mL is added in hole, maintains the concentration of Puromycin and MTX constant, 10 24 porocyte culture plates are put It returns in cell incubator, stationary culture 5 days.After 5 days, by the cell inoculation in 24 porocyte culture plates to 6 porocyte culture plates In, 2mL fresh cultures are added per hole, cell were passed in every 3 days, and passage ratio is 1:3, after carrying out 3 cell passages, We take a small amount of cell conditioned medium, are assessed the yield of fusion protein with the method for ELISA.According to ELISA as a result, choosing it The middle highest 10 plants of cells of fusion protein expression continue to expand and continue to cultivate in culture to 125mL cell shaking flasks, and finally select C6# plants of cell strains, which are determined, as seed cell is enlarged culture.

2.3 result

2.3.1Western Blot results

Cell behind 48 hours of the 1. transfection pCHO1.0- β-NGF-Fc plasmids of expression verification of Fig. 2 .1 β-NGF and IgG-Fc Supernatant；2. transfection β ,-NGF △ RRA-Fc plasmids cell conditioned medium after 48 hours；3. transfecting β-NGF △ opt-Fc plasmids 48 hours Cell conditioned medium afterwards；4. transfection pCHO1.0 plasmids cell conditioned medium after 48 hours.The cell suspension after transfecting 48 hours is taken to count, it will After cell density is adjusted unanimously, 1mL cell suspensions is taken to carry out the verification of destination protein expression respectively.1000rpm centrifuges 5min, Cell and cell conditioned medium are collected respectively, will be verified using Anti GAPDH antibody after cell cracking, cell conditioned medium makes respectively Expression verification is carried out with rabbit-anti humanβ-NGF antibody and mouse anti-human igg Fc antibody.Experimental result such as Fig. 2 .1, with transfection empty carrier CHO-S compare, after transfecting 3 kinds of purpose plasmids, can be clearly visible NGF and Fc expression, and have no related in negative control Ingredient.

2.3.2 ELISA experimental results

Fig. 2 .2 NGF content standard curve through WB it is experimentally confirmed that in cell conditioned medium really contain NGF and IgG1Fc ingredients, To further determine that the recombinant protein expression quantity contained in 48 hour cell supernatant of transient expression, this research using ELISA method into Measurement is gone.Because can not directly measure the content of fusion protein at present, we select Human β NGF ELISA Kit to measure cell The content of NGF in supernatant shows the content of fusion protein in cell conditioned medium indirectly.NGF content standard curves are shown in Fig. 2 .2, through surveying The content for determining NGF in cell conditioned medium is respectively：β-NGF-Fc：132ng/mL；

β-NGF△RRA-Fc：157ng/mL；β-NGF△opt–Fc：223ng/mL.

2.3.3 chick embryonic dorsal root ganglion measures fusion protein activity

Fig. 2 .3 chick embryonic dorsal root ganglions measure fusion protein activity Standard 1~5 and are followed successively by：3AU/mL standard items, 1AU/mL standard items, 0.33AU/mL standard items, 0.11AU/mL standard items, 0.03AU/mL standard items；Blank control is that transfection is empty 48 hour cell supernatant after carrier；β-NGF-Fc 1~5 are followed successively by：Dilute 45 times of cell conditioned mediums, dilution 135 times of cell conditioned mediums, Dilute 405 release 3645 times of a times cell conditioned medium, 1215 times of cell conditioned mediums of dilution, dilution cell conditioned mediums；β-NGF △ RRA-Fc are followed successively by： Dilute 50 times of cell conditioned mediums, 150 times of cell conditioned mediums of dilution, 450 times of cell conditioned mediums of dilution, 1350 times of cell conditioned mediums of dilution, dilution 4050 times of cell conditioned mediums；β-NGF △ opt-Fc 1~5 are followed successively by：70 times of cell conditioned mediums are diluted, 210 times of cell conditioned mediums is diluted, is dilute Release 5670 times of 630 times of cell conditioned mediums, 1890 times of cell conditioned mediums of dilution, dilution cell conditioned mediums.Neuromere is had not seen in negative control Growth illustrates not containing nerve growth factor in the cell conditioned medium of transfection empty carrier, and has transfected the cell of recombinant expression carrier In supernatant, it can be seen that apparent neural axon growth, illustrates that the CHO-S cells for having transfected recombinant expression carrier can be with table Up to β-NGF, and albumen is expressed with biological activity.By being compared with standard items, when sample concentration is 1ng/mL, in sample Ganglion axonal upgrowth situation is suitable with 1AU/mL in standard items, and the activity of judgement sample is respectively：β-NGF-Fc：135AU/ mL；β-NGF△RRA-Fc：150A U/mL；β -NGF△opt–Fc：210AU/mL.

2.3.4 first stage pressure is screened

By Fig. 2 .4 it is found that after MTX and Puromycin is added, cell viability continuously decreases, and the after screening starts Reach minimum within 10 days, about only 20% or so, subsequent cell viability starts to be gradually increasing, and the after starting screening the 23rd It, cell viability restores to 90% or more, indicates that the screening of first stage pressure terminates, cell enters second stage pressurized screen Choosing.

2.3.5 protein yield is assessed

In the latter stage of second stage pressure screening, we have carried out upgrowth situation to three plants of mixing clone cells and albumen produces The assessment of rate, by Fig. 2 .5 cell growth schematic diagrames.It is found that three plants of cell growth curves are almost the same, apparent too fast or mistake is had no Slow cell strain, and have Fig. 2 .6 protein yield schematic diagrames.It is found that the yield of cell strain its recombinant protein through codon optimization is bright Aobvious to be higher than remaining two plants of cell, β-NGF's in the 9th day our cell conditioned medium that measures can reach 15mg/L, and in addition The expressing quantity of two plants of cells only has 6mg/L or so, it is seen that have passed through the β-NGF △ opt-Fc cell strains after codon optimization Yield significantly improves, therefore the cell strain is selected to enter next step monoclonal screening stage.

2.3.6 monoclonal the selection result

25 piece of 96 porocyte culture plates is spread altogether in monoclonal screening stage, it is observed that monoclonal cell hole is obtained 527.By screening in 50 days, 10 plants of higher monoclonal cell strains of expression quantity are finally obtained, number is C#1~C# respectively 10.The assessment of expression quantity will be carried out after this 10 plants of cell expansion cultures again, assessment result is shown in Fig. 2 .7 difference clonal expressions Amount compares.10 plants of monoclonal cells are frozen, and cell strain C6# is taken to continue to expand culture for later-period purification and research.

2.4 discussing

In the first portion, by full genome synthesis and the method for over-lap PCR, recombined human β-NGF-Fc, β are obtained respectively Target gene is connected after double digestion and is expressed into pCHO1.0 by-NGF △ opt-Fc and β-NGF △ RRA-Fc gene orders In carrier, after identifying by bacterium colony PCR identifications, digestion digestion and identification be sequenced, sequencing result and objective gene sequence complete one It causes, illustrates successfully to construct purpose expression vector.But the expression vector of structure can give expression to desired destination protein actually, Expression quantity is how many, and whether the structure of destination protein is correct and actually either with or without biological activity, this just needs to carry out next The experiment of step.In terms of the selection of expression system, this research has been respectively compared escherichia expression system, insect and Yeast expression System and mammalian expression systems these three different expression systems.First, escherichia expression system is most commonly seen Expression system, it is a variety of that it has the advantages that expression quantity is high, expression speed is fast, low production cost, resistant to pollution ability are strong etc., It is the expression system of many recombination classes drug first choices.But Escherichia coli do not have endoplasm as a kind of prokaryotic expression system The organelles such as net, golgiosome cannot further modify the recombinant protein of generation, and come with regard to nerve growth factor Say, the folding of protein, glycosylation and disulfide bond formation, for the biological activity of nerve growth factor in vivo be all to Close important want.So although there are escherichia expression system above-described various advantages, this research to be also to give up selection Expression system of the system as our fusion proteins.Insect and yeast expression system are seen again, though the two is eukaryotic expression system System, but its glycosylated mode is not quite similar with mammalian expression systems again, and the nerve that we recombinantly express for guarantee is raw There is long factor fusion protein preferable biological activity, this research to have finally chosen mammalian expression systems in vivo.But It is that a variety of disadvantages, this research such as the yield of mammalian expression systems is relatively low, and speed of production is slower, and production cost is higher are also led to It is overpressurized screening and monoclonal and screens two processes, it is desirable to be stablized and height expresses the cell of nerve growth factor fusion protein Strain.This research selects CHO-S cell strains as our expression cell strain because the cell strain have the advantages that it is a variety of, first： CHO-S is a kind of mammalian cell, can promote the formation of disulfide bond, carries out accurate post translational processing, this includes egg The recombinant protein that glycosylation, carboxylated of white matter etc. make is either in terms of molecular structure, physicochemical property or biological function Closest to natural protein molecule；Second：CHO-S cells are a kind of fibroblasts, it seldom secretes the interior of itself Endogenous binding protein, the cell also have exocrine function, are conducive to the purifying of late-stage products；Third, CHO-S cells have higher Recombination expands and the ability of expression, helps to improve the expression quantity of recombinant protein；Finally, the cell adapted serum-free training It supports, the production in downstream is enormously simplified, but also production process is more prone to control.When transiently transfecting, the plasmid before transfection And cell preparation is particularly significant for transfection efficiency for improving.First, during plasmid extraction, it should follow strictly nothing Bacterium operates, and various vessel used and the first-class consumptive material of centrifuge tube, pipette, pipettor should be sterile, in plasmid extraction process In, to avoid the pollution of the inorganic salts such as organic solvent and sodium chloride, organic solvent pollution that cell can be caused dead as possible, nothing The pollution of machine salt can influence the formation of DNA- liposome compounds to influence transfection efficiency.In terms of cell preparation, select as possible The cell of the third generation is as transfection cell after recovery, and cell not centrifuge before transfection, otherwise can reduce transfection efficiency, to adopt Dilution method is taken to prepare cell suspension, cell viability when transfection need to be higher than 98%.Because transiently transfecting by cell state, transfection conditions Equal many factors influence, and transfection efficiency has very strong uncertain and expression quantity relatively low, so also needing to further experiment The cell strain of expression recombination fusion protein can be stablized by obtaining.Unlike transiently transfecting, in stable transfection, carry purposeful The expression plasmid of gene is entered by way of random integration in the genome of host cell, and can be with host cell Passage is replicated.But it is this integrate be a kind of chance event of small probability, still need using resistant gene to cell It is screened, to filter out because incorporating purpose carrier to obtain the positive colony of resistant gene.It should be noted that matter Grain is in the genome for being integrated into host cell it some times happens that imperfect integration, i.e. resistant gene have been integrated into genome And target gene is lost, and false positive clones are thus will appear, therefore also need to be the purpose clone that screening obtains Further screening.Because object of this investigation is to obtain the highest monoclonal cell of expression quantity, therefore selects the god in cell conditioned medium Content index as a filter through growth factor.During monoclonal screens, it is crucial that the first step, i.e., limited dilute Bed board this step is released, if dilution and plating procedure are appropriate, monoclonal hole count can be greatly increased.It should also be noted that in bed board, It has to selection to carry out when cell growth condition is good, can effectively improve the growth efficiency of monoclonal cell.

2.5 brief summary

CHO-S cells are transiently transfected by the method for liposome transfection, three plants are proved using the method for Western blot Clone cell equal successful expression destination protein is mixed, and the size of protein is consistent with expection；Using the method for ELISA into one Step demonstrates the successful expression of destination protein, and preliminary measurement has been done to expression quantity；Chick embryonic dorsal root ganglion method demonstrates table The fusion protein reached has biological activity, and activity is higher.So far, we can tentatively judge constructed by this research PCHO1.0- β-NGF-Fc, pCHO1.0 β-NGF △ opt-Fc and pCHO1.0 β-NGF △ tri- expression plasmids of RRA-Fc are equal Can be with high-efficiency transfection CHO-S cells, and molecular weight can be expressed correctly and cloned with the active three plants of mixing of good biological, Pressure screening by two stages and monoclonal screening, finally obtaining one plant can be with high efficient expression β-NGF-Fc fusion protein Cell strain is simultaneously named as CHO-NGF-Fc cell strains.

Purifying, physico-chemical analysis and its activity of fusion protein are surveyed

3.1 reagents and consumptive material

3.1.1 major experimental reagent

ProteinA affinity columns：HiTrapTM rProtein A FF are purchased from GE Healthcare；Ethyl acetate, Article No. 052-09041；12% triethylamine solution, article No. 200-20021；37% acetonitrile solution, article No. 018-26041；1- neoprenes Alkane, article No. 033-14371；PTH- amino acid eluents, article No. 168-27351；Trifluoroacetic acid, article No. 204-10771；25% 3 Fluoroacetic acid solution, article No. 201-10781；5% isothiocyanic acid benzoic acid solution, article No. 161-27341, PVDF membrane； Purchased from Japanese Shimadzu Corporation；The beginning of spring red colouring liquid is purchased from EnoGene companies, article No. E1WP306；Pierce BCA Protein Assay Kit：Purchased from Thermo fisher；Two hypophosphite monohydrate sodium dihydrogens, disodium hydrogen phosphate dodecahydrate, two citric acid monohydrates It is trisodium, citric acid, sodium hydroxide, sodium chloride, absolute ethyl alcohol, anhydrous calcium chloride, glacial acetic acid, potassium chloride, potassium dihydrogen phosphate, anhydrous Methanol is purchased from traditional Chinese medicines chemical reagents corporation；Tris-base is purchased from Sigma Aldriches；Furin protease, is purchased from New England Biolabs companies；1M HEPES Bhffer are purchased from life technologies companies；Serum-free DMEM Culture medium is purchased from GIBCO, article No. 12440；Fetal calf serum FBS is purchased from GIBCO, article No. 10099；Electrophoretic buffer：NuPAGE MES SDS Running Buffer (20X) are purchased from invitrogen companies, article No.：NP0007

Loading Buffer：NuPAGE LDS Sample Buffer (4X), it is public purchased from life technologies Department, article No.： NP0009；Sample reducing agent：NuPAGE Sample Reducing Agent (10X) are purchased from NOVEX companies, goods Number： NP0007

Pre-prepared colloid：4~12%Bis-Tris of NuPAGE Gel, 10Well；NuPAGE 10%Bis-Tris Gel, 10Well is purchased from invitrogen companies, article No. NP0322Box；Protein Marker：SeeBlue Plus2Prestained Standard is purchased from invitrogen companies, article No.：LC9259；Isoelectric Focusing Calibration Kit, purchase GE Healthcar company, article No. 17-0471；4-chloro-1-naphthol is purchased from Sigma companies；Two sulphur threoses Alcohol (DTT) is purchased from Sigma companies.

3.2 experimental method

3.2.1 the collection of cell conditioned medium

The C6# cells in exponential phase are collected, centrifugation is sowed after counting to the Tissue Culture Flask of 3 1000mL In, every bottle of addition 330mLCD Forti culture medium, after cell is put into cell incubator, 37 DEG C, 8%CO2,130~150rpm After continuous culture 5 days, all cell suspensions are collected.1000rpm centrifuges 10min, collects supernatant, is then centrifuged with low-temperature and high-speed Machine, 15000rpm, 4 DEG C of centrifugation 30min, cell fragment of leaving away collect whole supernatants, it is spare to 7.0,4 DEG C of refrigerations to adjust pH value.

3.2.2 protein A affinity chromatography

The preparation of mobile phase

Equilibration buffer A：Precision weighs two hypophosphite monohydrate sodium dihydrogen 1.37g, and disodium hydrogen phosphate dodecahydrate 4.37g adds Deionized water adjusts pH value to after 7.0 plus water is settled to 1000mL to 950mL.Use 0.45 μm of filtering membrane filtration, degassing After 15min, 4 DEG C spare, 6 months shelf-lifves.Elution buffer B：Precision weighs two citric acid monohydrate trisodiums 2.06g, citric acid 17.8g adds deionized water to 950mL, adjusts pH value to being settled to 1000mL with water after 3.0.Membrane filtration is filtered using 0.45 μm, It deaerates after 15min, 4 DEG C spare, 6 months shelf-lifves.Neutralization buffer：1.0M Tris-HCl adjust pH value to 9.0.Pillar is clear Washing lotion：Precision weighs sodium hydroxide 2.0g, and sodium chloride 58.5g is settled to 1000mL using deionized water, uses 0.45 μm of mistake Membrane filtration deaerates after 15min, and 4 DEG C spare, 6 months shelf-lifves.

Both it uses the equilibration buffer A of 10 column volumes to balance affinity column first, observes conductivity and baseline variation, wait for Tend to after stablizing, after reusing equilibration buffer A 5 column volumes of balance, prepares loading.Ready cell conditioned medium is taken out, is set In ice-water bath, it is 3ml per minute so that it is kept low temperature, setting loading speed.After completion of the sample, equilibration buffer is reused A rinses pillar, when observation conductivity and baseline turn again to the state of beginning, starts to elute albumen.Prepare sterile 1.5mL Centrifuge tube is several, and often 200 μ L of neutralization buffer are added in pipe, will be spare after centrifuge tube number consecutively.Mobile phase is converted at this time Elution buffer B, flow rate set is 3mL per minute, after starting elution, absorbing state at close observation UV detector 280nm Variation starts to collect efflux, each EP pipes are collected 0.5mL effluxes, arrived when 280nm goes out absorption value and starts significantly to increase Ultraviolet absorptivity stops collecting after being reduced to baseline at 280nm.It waits after eluting, is cleaned using the cleaning solution of 10 column volumes Pillar finally preserves pillar with 20% ethyl alcohol.

3.2.3 the reduction SDS-PAGE electrophoresis of purification of samples

Sample after purification, No. 1, No. 5, No. 10, No. 15, No. 20, No. 25, No. 30, No. 35, No. 40, No. 43 samples are taken respectively Reduction SDS-PAGE electroresis appraisals are carried out, electrophoresis method is shown in as follows：

The preparation of coomassie brilliant blue staining liquid：Weigh coomassie brilliant blue R250 1g, absolute methanol 400mL, absolute ethyl alcohol 500mL adds deionized water to 1000mL.The preparation of Coomassie brilliant blue destainer：Absolute methanol 300mL, glacial acetic acid 100mL are taken, Add deionized water to 1000mL.The preparation of sample before loading：20 μ L, Sample Reducing Agent of test sample, 2 μ L are taken, 5 μ L of LDS Sample Buffer amount to 27 μ L mixings, and boiling water bath is spare after 3 minutes.The preparation of electrophoretic buffer：Take MES SDS Running Buffer 25mL, it is spare to be settled to 500mL with deionized water.It tears pre-prepared colloid sealing strip off, carefully extracts sample Product are combed, and pre-prepared colloid is fitted into electrophoresis tank, above-mentioned electrophoretic buffer is filled in slot before and after electrophoresis tank, and 20 μ L samples are added per hole Product.Power on, first to start power supply under the conditions of 80V, after sample enters separation gel, voltage is turned up to 200V, until sample Product are run at the about 1cm of glue bottom, stop electrophoresis.

Gel after electrophoresis is taken out from slot, plastic shell is removed, gel is put into Coomassie brilliant blue dye liquor and dyes 1 It after hour, is put into destainer and decolourizes, change primary new destainer per hour, the transparence until gel background color becomes colorless stops Anti-avulsion color, gel is put into photographic system and is taken a picture.Electrophoresis result is shown in Fig. 3 .2, and according to electrophoresis result, 43 pipe purification of samples are closed And using the super filter tube 1000rpm of 10kD, 4 DEG C centrifuge 10min, after the redissolution of 400 μ L deionized waters is added, centrifuge, remove again Salt ion, up to purification of samples after being redissolved using 5mL deionized waters.

3.2.4BCA method measures the protein content of sample after purification

The preparation of standard curve：It is 2000 μ g/ to take the BSA standard protein solution water in kit to distinguish compound concentration The standard solution of mL, 1500 μ g/mL, 1000 μ g/mL, 800 μ g/mL, 600 μ g/mL, 400 μ g/mL, 200 μ g/mL.Sample Pre-dilution：Sample after purification is taken, dilutes 5 times, 10 times, 20 times and 40 times respectively with deionized water.The preparation of working solution：Take BCA Mixing after seminal plasma fructose detection kit solution A 49mL, reagent B solution 1mL, is protected from light spare.Sample-adding：Precision measure above-mentioned sample solution and Each 25 μ L of standard solution, are separately added into 96 clean porocyte culture plates, and a parallel multiple holes are arranged in each concentration. 200 μ L of working solution are added per hole, covers black cover board, gently vibrates 1min, after so that it is mixed well, 96 orifice plates are put into 37 DEG C In incubator, it is protected from light and is incubated half an hour.96 orifice plates are taken out after half an hour, are placed at room temperature for 5min, use suction at microplate reader detection 562nm Light value.Standard curve is drawn, the protein content in sample is calculated.

3.2.5 the Western Blot identifications of purification of samples

One, sample after purification is taken, 1mg/mL is diluted to deionized water, takes 20ul sample solutions, Loading is added 5 μ L, Sample Reducing Agent of Buffer, 2 μ L mixings, after boiling 2min, take 10 μ L loadings, primary antibody Rabbit Anti-Human NGF Antibody, the same 2.2.4 of experimental procedure

3.2.6 the N-terminal determined amino acid sequence of purification of samples

SDS-PAGE electrophoresis is restored, method is the same as 3.3.3. transferring films：Pvdf membrane after transferring film is put into vertical by method with 2.2.4. Spring red dye liquor takes out pvdf membrane and pvdf membrane is rinsed 2 to 3 times in ultra-pure water water, often at once when can be seen that apparent band Secondary 3 minutes, until film background color takes off completely.Destination protein band is cut, PPSQ-33A protein sequencers are put into, is transported 17 cycles of row.

3.2.7 the Furin endonuclease reactions of purification of samples

Reaction condition：25 DEG C of metal baths, digestion 6 hours

3.2.8 the Western Blot of sample are identified after digestion

Sample solution after 20ul digestions is taken, 5 μ L, Sample Reducing Agent of Loading Buffer, 2 μ L are added Mixing, after boiling 2min, it is Rabbit Anti-Human NGF Antibody to take 10 μ L loadings, primary antibody, and experimental procedure is same 2.2.4

3.2.9 after digestion sample it is secondarily purified

It learnt from else's experience the sample after Furin proteolytic cleavages, and carried out rProtein A affinity chromatographys again, obtain destination protein, It is measured through BCA methods and obtains fusion protein 2.02mg altogether.

3.2.10 uv scan

245nm~345nm length scannings are carried out to fusion protein using ultraviolet specrophotometer, scanning result is shown in figure

3.2.11 restoring SDS-PAGE measures molecular weight

Fusion protein is diluted to 1 μ g/mL, 10 μ L of sample volume, it is 20 μ L, electrophoresis side to add sample treatment liquid to total volume Method is shown in 3.3.3

3.2.12SDS-PAGE purity testing

Fusion protein is diluted to 1mg/mL, 10 μ L of sample volume, it is 20 μ L, electrophoresis side to add sample treatment liquid to total volume Method is shown in 3.3.3

3.2.13N- terminal amino acid sequence measures

The same 3.3.6 of experimental method

3.2.14 isoelectric focusing electrophoresis surveys fusion protein isoelectric point

Sample treatment：Fusion protein is diluted to 1mg/mL, takes the spare prerunnings of 5 μ L about 5 minutes, is stained with using sample comb Take a small amount of sample loading.Electrophoresis：Under voltage stabilizing, 100V starts to focus.Electrophoresis increases voltage to 200V, electrophoresis 15 after 15 minutes Voltage is further added by 450V after minute, stops voltage when electric current drops to 1mA.Dyeing：Gel is taken out, fixer (trichlorine Acetic acid) in fix half an hour, gel is transferred to dyeing 1 hour in dyeing liquor (silver nitrate solution), then will be after dyeing it is solidifying Glue, which is put into destainer, to decolourize until gel clear background.

3.2.15 identification experiment (Western blot)

Sample treatment：Fusion protein is diluted to 1 μ g/ μ L, takes 10 μ L loadings.Take mNGF as on 10 μ L of positive control Sample.SDS-PAGE electrophoresis and transferring film：With Western Blot.Primary antibody is incubated：Pvdf membrane after transferring film is put into plate, is added PBS buffer solution 15mL, 30 μ L of rabbit-anti humanβ-NGF antibody, are eventually adding the drop of horse serum 5, and room temperature is rocked overnight.PBS buffer solution is washed Film 4 times, 2 minutes every time.Secondary antibody is incubated：After washing film, secondary antibody 5 in PBS buffer solution 15mL, ABC-6200Kit is added into plate Drop, horse serum 5 drip, and room temperature rocks incubation within 1 hour.PBS buffer solution washes film 4 times, 2 minutes every time.Amplification agent is added into plate (each 5 drop mixing of A liquid, B liquid, need to shift to an earlier date 1 hour and prepare in kit), room temperature rocks incubation 1 hour.PBS buffer solution washes film 4 It is secondary, 2 minutes every time.Colour developing：Developing solution is prepared：15mL absolute methanols+color developing agent (4-chloro-1-naphthol, chloronaphthol) + 15 μ L hydrogen peroxide mixings a little, pvdf membrane is put into developing solution and is rocked, color development stopping when apparent band occurs.

3.2.16 mass spectroscopy molecular measures fixed.

The processing of test solution takes 50 μ L test samples (1mg/mL), the DTT solution of 2 μ L 1mol/L is added, 50 DEG C incubate 1h is educated, it is spare.Ultra performance liquid chromatography parameter setting, column temperature are 35 DEG C；Sample storage temperature is 10 DEG C；10 μ g of applied sample amount；It presses Following gradient elution：

Mass spectrometer parameters are set, Mass Spectrometry Conditions：MS type collection data；Capillary voltage：3000V；Cone voltages：40V； Go solvent gas temperature：350℃；Source temperature：120℃；Remove solvent gas flow velocity：800L/h, scanning range (m/z)：500-3000. Molecular weight determination is corrected instrument in scanning range with prepared mass spectrograph calibration solution, connects liquid chromatograph With mass spectrograph, the measurement of molecular weight is carried out by the program of setting.

3.2.17 Mass Spectrometric Identification after enzymolysis.

Fusion protein is diluted to 1mg/mL, 10 μ L of sample volume carry out reduction SDS-PAGE electrophoresis, and electrophoresis method is shown in 3.3.3.Gel after electrophoresis is put into Coomassie brilliant blue dye liquor and dyes 1h, gel after dyeing is put into destainer until background Color is sloughed.The blob of viscose for needing to analyze is cut, is put into clean 1.5mL centrifuge tubes, the deionization of 1mL is added into centrifuge tube Water cleans 10min, water is removed, and repeats the above steps primary.The in-gel digestion destainer of 1mL is added into centrifuge tube, clearly 10min is washed, destainer is removed, is repeated once.In-gel digestion destainer configures：Second is added in 50% acetonitrile, 25mM ammonium hydrogen carbonate Nitrile is dehydrated to micelle to bleach completely, and vacuum drains acetonitrile.Add 10mM DTT, allow micelle to absorb complete, be put into 56 degree of water-baths, It is incubated 1 hour.After incubation, extra DTT liquid is removed, 55mM IAM are added, darkroom is incubated at room temperature, 45 minutes.It is incubated After, extra IAM liquid is removed, 25mM ammonium hydrogen carbonate is added, is cleaned 10 minutes, and repeated washing is primary.Remove bicarbonate Ammonium is added destainer and cleans 10 minutes, and is repeated once.Acetonitrile is dehydrated to micelle to bleach completely, and vacuum drains acetonitrile. 1μg/μ The enzyme liquid storage of L dilutes 15 times with 25mM ammonium hydrogen carbonate, is added in dewatered micelle, micelle is allowed to fully absorb.Then it is added 25mM ammonium hydrogen carbonate did not had micelle, was put into 37 degree of water-baths, digestion is overnight.Machine on 10 μ L samples is taken, is detected using mass spectrograph, liquid Phase gradient and mobile phase：Liquid phase：prominence nano 2D(shimazhu)

Column material：C18,5um, 150A (Eprogen)；Flow velocity：400nl/min；Liquid phase gradient：See Fig. 3 .19 liquid phase gradient tables And curve graph

Mass spectrometer：MicrOTOF-QII(BrukerDaltonics)

Data acquisition software：BrukerDaltonicsmicrOTOFcontrol

MS/MS scanning ranges：50-2200m/z

Collision gas：Argon gas

Capillary voltage：1500V

Dry gas temperature：150℃

3.2.18 chick embryonic dorsal root ganglion measures fusion protein activity.

By 200 times of mouse nerve growth factor Bioassay reference pre-dilution, × 1, × 3, × 9, × 21 is then carried out respectively, × Determination of activity is carried out after 81 times of dilutions.By without 100000 times of the fusion protein pre-dilution of Furin protease digestions, then Determination of activity is carried out after carrying out × 1, × 3, × 9, × 21, × 81 times of dilutions respectively.By the fusion Jing Guo Furin protease digestions 70000 times of albumen pre-dilution carries out determination of activity after then carrying out × 1, × 3, × 9, × 21, × 81 times of dilutions respectively.It uses DMEN culture mediums are negative control

3.2.19TF-1 cell/MTS colorimetric method for determining NGF activities.

The preparation of reagent.

Basal medium：It measures fetal calf serum (FBS) 50mL to be added in PRMI1640 culture solutions 450mL, 4 DEG C of preservations.It is complete Full culture medium：Mouse nerve growth factor mNGF (Soviet Union's peptide life, SHUTAISHEN Pharmacy stock Co., Ltd) is added in basal medium Contain mNGF12U per 1mL to final concentration of.PBS buffer solution：Weigh disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, sodium chloride 8g, potassium chloride 0.2g are dissolved in water to 1L, and through high pressure sterilization, the room temperature preservation time limit is no more than 12 months.

The preparation of TF-1 cells.

TF-1 cell strains complete medium is cultivated in 37 DEG C, 5% carbon dioxide cell incubator, controls cell concentration Contain 1.0 × 105~5.0 × 105 cells in 1ml culture solutions, is used for NGF Determination of biological activity within 24 hours after passage.

Experimental method

Preparatory work of experiment：Culture medium will be analyzed, PBS buffer solution is preheated to 37 DEG C, and MTS is protected from light dissolving, and it is spare to set 4 DEG C of refrigerators.

It is prepared by TF-1 cell suspensions：The TF-1 cell suspensions of the amount of taking fully exponential phase move in 50mL centrifuge tubes, 1000rpm is centrifuged 5 minutes, discards supernatant liquid, collects TF-1 cells.After cell is resuspended addition 15mLPBS buffer solutions, again 1000rpm is centrifuged 5 minutes.Above-mentioned steps are repeated 2 times, and cell, which is formulated as every 1mL, with basal medium contains 5 × 104 carefully The cell suspension of born of the same parents, sets 37 DEG C, spare in 5% carbon dioxide cell incubator.

The preparation of standard solution：After taking recombinant human nerve growth factor standard items to redissolve, with basal medium pre-dilution To 100U/mL.The preparation of test solution：After recombined human β-NGF-Fc fusion proteins are dissolved, with basal medium pre-dilution To 100U/mL (often step dilution is no more than 10 times).Prepare sample gradient：In 96 porocyte culture plates, successively from A rows to H rows 3 times of doubling dilutions are carried out, totally 8 dilutions, each dilution do 2 holes, and each hole leaves 100 μ L solution, discards hole respectively Middle redundant solution, it is spare.100 holes μ L/ cell suspensions are added into above-mentioned 96 porocyte culture plates, is put into wet box, is placed in 37 DEG C, it is continuously cultivated in 5% carbon dioxide cell incubator 72 hours.After 72 hours, 20 μ L MTS solution are added into every hole, It puts back in cell incubator and continues culture 3 hours.As a result it measures：After 3 hours, tissue culture plate is vibrated 2 minutes, enzyme mark is put into Instrument goes out in wavelength 490nm and measures absorbance, records measurement result.

3.3 result.

3.3.1 protein A affinity chromatography result.

Cell conditioned medium is through ProteinA affinitive layer purification destination proteins, by Fig. 3 .1 it is found that we only receive in elution stage Collect an eluting peak, obtains 43 1.5mL centrifuge tubes of purifying protein altogether.It selects wherein 10 samples and carries out reduction SDS- PAGE electrophoresis, electrophoresis result are shown in Fig. 3 .2, and two eggs are obtained altogether near 50kD and 60kD through SPS-PAGE reduction electrophoresis discoveries The molecular weight of informal voucher band, growth factor of human nerve is about 13kD, and IgG1Fc sections of molecular weight is about 34kD, judges that destination protein is answered 50kD or so should be located at, and albumen is present in all stages of eluting peak efflux at 60kD, and illustrate that the albumen can be with ProteinA has high-affinity, and albumen should also contain Fc sections at this.To further determine that, what band is at 60kD, this Fusion protein is identified in research and utilization WB and -terminal amino acid sequencing.

3.3 result

3.3.1 protein A affinity chromatography result

Cell conditioned medium is through ProteinA affinitive layer purification destination proteins, by Fig. 3 .1 it is found that we only receive in elution stage Collect an eluting peak, obtains 43 1.5mL centrifuge tubes of purifying protein altogether.It selects wherein 10 samples and carries out reduction SDS- PAGE electrophoresis, electrophoresis result are shown in the reduction SDS-PAGE electrophoresis of Fig. 3 .2 affinity purification products, and M is protein Marker, 1~ No. 11 samples are respectively No. 1, No. 5, No. 10, No. 15, No. 20, No. 25, No. 30, No. 35, No. 40, No. 43 samples, through SPS-PAGE Reduction electrophoresis discovery obtains two protein bands altogether near 50kD and 60kD, and the molecular weight of growth factor of human nerve is about 13kD, IgG1Fc sections of molecular weight is about 34kD, judges that destination protein should be located at 50kD or so, and albumen is present at 60kD All stages of eluting peak efflux illustrate that the albumen can have high-affinity with ProteinA, and albumen should also contain at this There are Fc sections.To further determine that, what band is at 60kD, this research and utilization WB and -terminal amino acid sequencing are to fusion protein It is identified.

3.3.2 BCA methods measure the content of purified product.

BCA methods measure albumen concentration standard curve and see Fig. 3 .1, according to a concentration of of standard curve determination purifying protein 4.69mg/mL obtains fusion protein 23.45mg altogether.

3.3.3 the Western Blot qualification results of fusion protein.

Western Blot identification experiment results are shown in Fig. 3 .2, and the albumen of the vicinity 50kD and 60kD can be with as seen from the figure It is combined with anti-ngf antibodies, illustrates that albumen contains NGF ingredients at two.The Western Blot identifications of Fig. 3 .3 purified products.M: Protein Marker；1~4：β-NGF-Fc fusion protein samples.

3.3.4 the -terminal amino acid sequencing results of fusion protein

Fig. 3 .4 are the N-terminal sequencing result of protein band at 50kD, and the amino acid sequence measured is：1.Ser；2.Ser； 3.Ser； 4.His；5.Pro；6.Ile；7.Phe；8.His；9.Arg；10.Gly；11.Glu；12.Phe；13.Ser； 14.Val； 15.Cys；It is completely the same with 15 amino acid sequences before NGF through comparing, judge that band melts for β-NGF-Fc at 50kD Hop protein.Fig. 3 .5 are protein band N-terminal sequencing result at 60kD, and the amino acid sequence measured is：1.Glu；2.Pro； 3.His； 4.Ser；5.Glu；6.Ser；7.Asn；8.Val；9.Pro；10.Ala；11.Gly；12.His；13.Thr； 14.Ile； 15.Pro；It is completely the same through comparing preceding 15 amino acid sequences of protein band and proNGF at this, judge 60kD Place's protein band is proNGF-Fc fusion proteins.Protein band N-terminal sequencing result at Fig. 3 .5 60kD.

3.3.5 Western Blot qualification results after the digestion of Furin protease

Digestion rear fusion protein Western Blot identification experiment results are shown in Western after Fig. 3 .6Furin protease digestions Blot qualification results M：Protein Marker；1:Fusion protein sample before digestion；2：Furin protease digestion rear fusion proteins, As seen from the figure after Furin digestions, the NGF precursors at 60kD are cut off.

3.3.6 fusion protein uv scan result

245nm~345nm uv scans are carried out to fusion protein, result figure is shown in Fig. 3 .7, as seen from the figure recombined human β-NGF-Fc fusion proteins have maximum absorption band at 280nm.

3.3.7 reduction SDS-PAGE measures molecular weight results

The reduction SDS-PAGE molecular weight determinations of sample are shown in Fig. 3 .7, and wherein 1-11 bands are Marker bands, band S is recombinant beta-NGF-Fc fusion proteins, and the fusion protein molecule amount known to result is about 47.65kD.

3.3.8SDS-PAGE fusion protein purity is measured.

SDS-PAGE purity testing results are shown in that Fig. 3 .8SDS-PAGE measure fusion protein purity, and PAGE glue is through gel imaging System scanning shares three bands, wherein No. 3 are purpose band, accounts for the 93.39% of all bands.That is the purity of fusion protein is 93.39%.

3.3.11 identification experiment (Western blot) result.

Western blot identification experiment result is shown in Fig. 3 .11, and band 1,2 is recombination humanβ-NGF's-Fc fusion proteins, item in figure Band M is protein pre-dyed marker, and band 3 is that mouse lower jaw gland extracts NGF.Fusion protein can be with rabbit-anti humanβ-NGF Dan Ke Grand antibody specificity combines.

3.2.12 mass spectroscopy fusion protein molecule amount result

Mass spectrometric determination fusion protein molecule amount result is shown in that Fig. 3 .12 and 3.13, wherein Fig. 3 .12 are fusion protein multi-charge Mass spectrogram, Fig. 3 .13 are molecular weight determination.The theoretical molecular weight of recombined human β-NGF-Fc fusion proteins is 39472.96Da, At Fc sections, fucose, the theoretical molecular weight of theoretical molecular weight 1444.53Da, i.e. fusion protein are containing there are one 40917.49Da, mass spectrum actual measurement molecular weight is 40917.20Da known to result, and the two differs 0.29Da, and relative error is 0.0007%.

3.3.13 the Mass Spectrometric Identification of fusion protein.

Using Mass Spectrometric Identification is done after trypsin hydrolysis β-NGF-Fc fusion proteins, 22 peptide fragments and theoretical peptide fragment are obtained To matching (table 1), total amino acid coverage rate is 77%.The parts wherein β-NGF are obtained 9 peptide fragments and are consistent with theoretical peptide fragment, ammonia Base acid coverage rate is 78.3%.The parts IgG1Fc are obtained 13 peptide fragments and are consistent with theoretical peptide fragment, and amino acid coverage rate is 76.7%.

Table one：The peptide fragment to match with theoretical sequence and position.

Fixed modifications：Carbamidomethyl(C)

Variable modifications：Gln->Pyro-Glu (N-term Q), Oxidation (M)

Cleavage by Trypsin：cuts C-term side of KR unless next residue is P

Sequence Coverage：77%

Matched peptides shown in Bold Red

3.3.14 chick embryonic dorsal root ganglion method measures fusion protein activity

Table two：Chick embryonic dorsal root ganglion method determination of activity result

Fig. 3 .15 chick embryonic dorsal root ganglion methods measure fusion protein activity.

After measured, it is 1.04 × 105AU/mg that the ratio of the fusion protein after Furin protease digestions, which is lived, without enzyme It is 6.54 × 104AU/mg that the ratio for the fusion protein cut, which is lived,.

3.3.15TF-1 cell/MTS colorimetric method for determining NGF activities.

Fig. 3 .16 are one of five determination of activity results, by taking the secondary result as an example：3 curves, wherein grey are shared in figure Curve is standard curve, and blue curve is the fusion protein after Furin protease digestions, and red curve is without passing through enzyme The fusion protein cut passes through by figure below it is found that regardless of whether the fusion protein by digestion can stimulate the proliferation of TF-1 cells Fitting can obtain typical quadruplex parameters, and R2 is all higher than 0.99.

According to calculation formula：

The activity for measuring digestion rear fusion protein is 3.6 × 105U/mL, and the activity without the fusion protein of digestion is 5.02× 105U/mL.The albumen concentration measured according to BCA methods:Digestion rear fusion protein is 2.02mg/mL；Without digestion Fusion protein is 4.69mg/mL, and the ratio that digestion rear fusion protein is obtained according to specific activity calculation formula is lived as 1.84 × 105U/ Mg, it is 1.07 × 105U/mg to live without the ratio of the fusion protein of digestion.It can be seen that the fusion after Furin protease digestions The ratio work of albumen significantly improves, and thus infers that proNGF precursors expose free N-terminal after Furin protease digestions, this When NGF just have biological activity.Five experiments measure result and summarize and see the table below：

Table three：TF-1 cells/MTS colorimetric method determination of activity results

3.4 discussing

ProteinA affinity chromatographys are the most frequently used methods to purify the albumen containing IgG structures.The ring for being 7.0 in pH value In border, the CH2 that ProteinA can be with Fc sections, CH3 mechanisms domain specific binding, to detach destination protein with foreign protein. ProteinA affinity chromatographys are easy to operate, and the ProteinA fillers per 1mL can be in conjunction with the Fc section albumen of 50mg, protein recovery It is higher, it is not only suitable for the purifying and research of a small amount of albumen, large-scale production is also applied for and prepares.Rear fusion protein is purified through BCA Method measured concentration is 4.69mg/mL, obtains fusion protein 23.45mg altogether.Fusion protein is found after restoring SDS-PAGE electrophoresis There are two protein bands at 50kD and 60kD respectively.Inferred according to molecular weight, the molecular weight of β-NGF-Fc fusion proteins should In 48kD or so, so infer that the protein band at the places 50kD should be purpose band, but the protein band at 60kD is present in and washes De- peak so in the stage, illustrate that the albumen should not be the foreign protein with ProteinA non-specific bindings, but also contain Fc Segment with ProteinA to specifically bind.In order to further determine the ingredient of the two protein bands, this research uses rabbit Anti-human β NGF antibodies have carried out WB identifications to fusion protein, as a result show that albumen can be anti-with rabbit-anti people β NGF at 50kD and 60kD Body is specifically bound, it was demonstrated that albumen also has NGF ingredients at 60kD.In view of the two separating degree is preferable, we are by destination protein After electrophoretic separation, cuts carried out -terminal amino acid sequencing respectively.Preceding 15 amino of albumen at 50kD after measured Acid is respectively：SSSHPIFHRGEFSVC, it is completely the same with preceding 15 amino acid of NGF, it was demonstrated that albumen is β-NGF- really at this Fc fusion proteins.And preceding 15 amino acid of the albumen at 60kD is respectively：15 before EPHSESNVPAGHTIP, with proNGF A amino acid is completely the same, it was demonstrated that albumen is without the NGF precursor proteins Jing Guo digestion herein.In vivo, complete NGF Exon is made of 241 amino acid, normally referred to as preproNGF, preproNGF precursors preceding 18 ammonia in endoplasmic reticulum The signal peptide of base acid is cut, and forms proNGF precursors, and proNGF precursors contain 223 amino acid, in endoplasmic reticulum Exist in the form of homodimer, is then transferred in golgiosome, Furin protease can be with specific recognition herein The sites Arg-Ser-Lys-Arg in proNGF precursors are cut, and are generated and are transported to after ripe NGF extracellular, while also having The not cut proNGF precursors of small part are secreted into extracellular.This research purifying has obtained a large amount of not cut proNGF Precursor protein considers whether to be likely to be the Apoptosis in cell cultivation process and discharges a large amount of intracellular proteins after cracking, to So that proNGF precursors is entered in cell conditioned medium, but this experiment strictly controls incubation time in cell culture, collects cell Cell viability is up to 98% when supernatant, and the above situation would not occur.And it in the WB experiments after transient transfection and has not seen Band at 60kD, it is relatively low that consideration should be proNGF contents, fails to be detected discovery.It is mould in the MTX and purine in follow-up two stages After element pressurization screening and monoclonal screening, the higher cell strain of expression quantity is obtained, makes endogenic Furin protease relatively not Foot, cannot cut proNGF precursors completely so that a large amount of proNGF precursors have been released to extracellular.Because of proNGF precursors Also contain Fc segments, therefore also come out by ProteinA affinitive layer purifications.Then this experiment is right in vitro using Furin protease Fusion protein has carried out digestion, and the proNGF precursors after WB identifies digestion at 60kD are removed, and have obtained single purpose item Band.

3.5 brief summary

Destination protein is obtained using rProteinA affinitive layer purifications, fusion protein is obtained altogether through BCA methods measurement 23.45mg has found that there are two purpose bands at 50kD and 60kD respectively through restoring SDS-PAGE electrophoresis.Utilize the ends WB and N- Two protein bands of amino acid assays pair are measured, and identified two purpose bands can be with rabbit-anti humanβ-NGF's antibody knot It closes, preceding 15 amino acid sequences of protein band are at 50kD：SSSHPIFHRGEFSVC, it is complete with preceding 15 amino acid of NGF It is complete consistent.Preceding 15 amino acid of albumen at 60kD is respectively：EPHSESNVPAGHTIP, preceding 15 amino with proNGF Acid is completely the same, it was demonstrated that protein band is NGF precursors at this.Digestion is carried out to fusion protein in vitro using Furin protease Afterwards, precursor protein is cut out at 60kD, and single destination protein is obtained after secondarily purified.SDS-PAGE electrophoretic determinations melt The purity of hop protein is 93.39%；The molecular weight for restoring SDS-PAGE electrophoretic determination fusion proteins is 47.65kD；Ultraviolet spectra Scanning fusion protein has maximum absorption band at 280nm；Isoelectric focusing electrophoresis measure fusion protein isoelectric point be 4.55~ 5.85；Digestion rear fusion protein N-terminal amino acid sequencing result be SSSHPIFHRGEFSVC, N-terminal it is uniform and with 15 before NGF Amino terminal sequence is completely the same；Identification experiment (Western blot) identifies that fusion protein can be with rabbit-anti people's β NGF monoclonal antibodies Specific binding；The molecular weight of mass spectrometric determination fusion protein is 40917.49Da；Fusion protein is laggard through trypsin digestion Row Mass Spectrometric Identification obtains 22 protein fragments being consistent with theoretical peptide fragment altogether, and amino acid coverage rate is 77%；Chicken embryo Dorsal root god The ratio that warp knuckle method measures digestion rear fusion protein is lived as 1.04 × 105AU/mg, and the ratio work without the fusion protein of digestion is 6.54×104AU/mg；The ratio work that TF-1 cells/MTS colorimetric methods measure digestion rear fusion protein is 1.84 × 105U/mg, not It is 1.07 × 105U/m that the ratio of fusion protein by digestion, which is lived,.

Recombinant cell strain is examined and determine.

4.1 experiment reagents and instrument

4.1.1 experiment reagent

Serum free medium CD Forti CHO are purchased from Life technologies companies；The agent of resistive connection group, Anti- Clumping Agent are purchased from gibco companies；Glutamine, Glutamine are purchased from gibco companies；Fetal calf serum, FBS, purchase From gibco companies；MEM culture mediums are purchased from Life technologies companies；0.25% pancreas enzyme -EDTA, it is public purchased from gibco Department；Dibenzamide fluorescent dye is purchased from Sigma companies；Absolute methanol, glacial acetic acid, citric acid, disodium hydrogen phosphate, glycerine are equal Purchased from traditional Chinese medicines chemical reagents corporation.

4.2 experimental method

4.2.1CHO-NGF-Fc cellular morphology

CHO-NGF-Fc and CHO-S cells are taken to be inoculated in 75cm with 5 × 105/mL density²In Tissue Culture Flask, every bottle connects Kind 15mL.Tissue Culture Flask is placed in 37 DEG C, 8%CO2, is cultivated 3 days in the cell incubator of humidity 80%.After three days, cell When growing to 70%~80% and converging, film recording cellular morphology.

4.2.2CHO-NGF-Fc cell virus factors check.

1.0 × 105/mL of BHK-21 cells in good condition is taken to be inoculated in 25cm²Tissue Culture Flask, addition MEM culture mediums+ The total 7mL of 3% fetal calf serum is cultivated 24 hours under 37 DEG C, 5% carbon dioxide conditions, makes its abundant adherent growth.CHO-NGF- Continuously culture takes cell suspension to Fc after three days, multigelation cell 3 times (- 70 DEG C freeze 1 hour, and 37 DEG C melt 30 minutes), 1000rpm is centrifuged 5 minutes, takes supernatant 0.5mL to be added in the BHK-21 cells of culture 24 hours, 37 DEG C, 5% carbon dioxide Under the conditions of cultivate 7 days, while doing negative control with MEM culture solutions 0.5mL.After culture 7 days, cell state, multigelation are observed Cell 3 times (- 70 DEG C freeze 1 hour, and 37 DEG C melt 30 minutes), 1000rpm is centrifuged 5 minutes, collects supernatant.It takes in good condition 1.0 × 105/mL of BHK-21 cells is inoculated in 25cm²Tissue Culture Flask, be added+3% total 7mL of fetal calf serum of MEM culture mediums in 37 DEG C, cultivate 24 hours under 5% carbon dioxide conditions, make its abundant adherent growth.It is inhaled after 24 hours and abandons culture solution 3mL, be added 3rd step collects supernatant 3mL.37 DEG C, cultivate 7 days under 5% carbon dioxide conditions.After culture 7 days, cell state is observed.Repeat 3,4 Step is primary.MEM culture solutions are supplemented according to cell state in incubation.After culture 7 days, cell state is observed.

4.2.3 different generation CHO-NGF-Fc cell strain expression quantity measure.

The third generation is reached after taking P5, P10, P15, P20, P25, P30, P35, P40 and P45 to recover for recombinant cell strain respectively Shi Lianxu takes cell conditioned medium after cultivating five days, and fusion protein expression is measured using ELISA method.

4.2.4CHO-NGF-Fc cell detection of mycoplasma.

The preparation of cell culture.After cell to be measured is passed on three times using antibiotic-free culture medium, continuous culture three days with On do not change liquid, take cell suspension 1000rpm centrifuge 5 minutes, take cell conditioned medium spare in 4 DEG C.Indicator cells (Vero cells) Product.The Vero mono- for being proved no mycoplasma contamination is taken, in 25cm after recovery²In Tissue Culture Flask, antibiotic-free is added The total 5mL of+5% fetal calf serum of DMEM culture mediums is placed in 37 DEG C, is cultivated under 5% carbon dioxide conditions, is carried out when cell covers with thin Born of the same parents are passed on, and cell at least passes on primary rear use.Counting is centrifuged after taking Vero cell dissociations before experiment, it is close with 1.0 × 105/mL Degree reaches 25cm²In Tissue Culture Flask, 37 DEG C, 5% carbon dioxide conditions overnight incubation.Sample to be tested is inoculated with, and is taken and is prepared Cell culture 2mL is added in Vero cells, using the DMEM of antibiotic-free as negative control, is added in 2mL and Tissue Culture Flask, Cell is put back in cell incubator after inoculation and is cultivated, indicator cells at least pass on once, when last passes on extremely by cell inoculation It is cultivated 5 days in 6 orifice plates containing coverslip.DNA is dyed and detection, is sucked out in the liquid and 6 orifice plates in culture plate, adds after 5 days Entering fixer, (absolute methanol is with glacial acetic acid by 3:1 mixing, matching while using) 5mL, 5 minutes are placed at room temperature for, whole fixers are sucked out. Fresh fixative 5mL is added again, is placed at room temperature for 10 minutes, all fixers is sucked out, be that coverslip spontaneously dries in air. Dibenzamide fluorescent dye work also 5mL is added per hole, closes the lid, is placed at room temperature for half an hour.Dyeing liquor in hole is sucked out, 5mL sterile water for injection is added per hole to wash 3 times, excessive moisture is sucked out with filter paper, coverslip is in air drying.Take cleaning Glass slide simultaneously drips mounting liquid and (takes 0.1mol/L liquor sodii citratis 22.2mL, 0.2mol/L disodium phosphate soln 27.8mL, take Glycerine 50mL mixings adjust pH value to 5.5) 1 drop, coverslip are faced down and is covered on mounting liquid, mounting is made, is fallen with fluorescence Set microscopically observation.

4.3 result

4.3.1 the form of CHO-NGF-Fc cells

By Fig. 4 .1 it is found that CHO-NGF-FC cellular morphologies are rounded, suspension growth.The form both compared with CHO-S cells It is similar, no significant difference.

4.3.2 the detection of different generation monoclonal cell express express target protein amounts.

It takes the monoclonal cell of different generations to recover respectively, reaches continuously to cultivate 5 days after the third generation cell conditioned medium is taken to carry out The detection of NGF contents.Detection expression quantity is shown in Fig. 4 .2, P5~P45：Different generation CHO-NGF-Fc cell strains as seen from the figure, cell Reach 45 generation NGF expression quantity no significant differences, about 18mg/L.Illustrate still stablize expression mesh when cell is passaged to 45 band Albumen, do not decay.

4.3.3 CHO-NGF-Fc cell virus factors checks.

After viral sensitive cells virus infection, apparent cytopathy often occurs.BHK-21 cells are to detach since the childhood The single cell clone cell of hamster,syrian kidney, for viral sensitive cells.The cell is fibroblast, in regular culture conditions Lower adherent growth has infected shrinkage after susceptible virus and has been rounded to rupturing, fall off.And NGF to the growth of BHK-21 cells without promotion Effect, therefore for detecting pollution of the cell strain with the presence or absence of virus, by Fig. 4 .3 experimental group BHK-21 cells pictures (X100), 4.4 After control group BHK-21 cells picture (X100) is as it can be seen that cultivate 21 days, experimental group cell normal growth, no lesion, with control group Cell growth condition is identical.

4.3.4 CHO-NGF-Fc detection of mycoplasma.

Verification result is shown in .5~4.6 Fig. 4, in negative control group, only it can be seen that the nucleus of indicator cells shows Huang Green fluorescence is observed that fluorescent color particle differ in size, irregular in positive control, under fluorescence microscope. CHO-NGF-Fc cell strains are in yellow green in microscopic observation visible cell core, and extracellular no irregular particle shape or Filamentous fluorescence Color.Prove the cell strain without mycoplasma contamination.

4.4 discussing

This research has carried out this plant of monoclonal cell preliminary inspection after the structure for completing CHO-NGF-Fc cell strains Fixed work.The result shows that the recombinant cell strain of structure is morphologically rounded, suspension growth.With the CHO-S for not doing any transfection Cell is compared, and the two form is similar, has no apparent morphological change.The inspection of NGF expression quantity has been carried out to multiple passage cell It surveys, the experimental results showed that cell, which was passaged to for 45 generations, can still stablize expression recombinant human nerve growth factor, and expression quantity is not sent out Raw decaying.Using BHK-21 virus sensitive cells detection CHO-NGF-Fc cell strains, whether there is or not virokine infection, the results showed that should The virus-free factor infection of recombinant cell strain, using indicator cells method detection CHO-NGF-Fc cell strains, whether there is or not mycoplasma infection, knots Fruit shows the cell strain without mycoplasma contamination.But also many detection projects need subsequently to be carried out, for example extraction cell is total It carries out whether RT-PCR detection gene transcription levels are changed after RNA, detects recombination in cell using Q-PCR and copy Whether shellfish number changes.

Sequence table

<110>National Institute for Food and Drugs Control

<120>The Chinese hamster ovary celI strain of efficiently expressing recombinant human β-NGF-Fc fusion proteins and its construction method

<150> CN2016108157382

<151> 2016-09-12

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 725

<212> DNA

<213> pCHO1.0-beta-NGF-Fc

<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

cgctttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720

gcctg 725

<210> 2

<211> 725

<212> DNA

<213> pCHO1.0-beta-NGF-opt-Fc

<400> 2

atgagcatgc tgttctacac cctgatcacc gccttcctga tcggcataca ggccgagccc 60

cacagcgaga gcaacgtgcc cgccggccac accatccccc aggcccactg gaccaagctg 120

cagcacagcc tggacaccgc cctgcgccgc gcccgcagcg cccccgccgc cgccatagcc 180

gcccgcgtgg ccggccagac ccgcaacatt accgtggacc cccgcctgtt caagaagcgc 240

cgcctgcgca gcccccgcgt gctgttcagc acccagcccc cccgcgaggc cgccgacacc 300

caggacctgg acttcgaggt gggcggcgcc gcccccttca accgcaccca ccgcagcaag 360

cgcagcagca gccaccccat tttccaccgc ggcgagttca gcgtgtgcga cagcgtgagc 420

gtgtgggtgg gcgacaagac caccgccacc gacatcaagg gcaaggaggt gatggtgctg 480

ggcgaggtga acattaacaa cagcgtgttc aagcagtact tcttcgagac caagtgccgc 540

gaccccaacc ccgtggacag cggctgccgc cgcattgaca gcaagcactg gaacagctac 600

tgcaccacca cccacacctt cgtgaaggcc ctgaccatgg acggcaagca ggccgcctgg 660

cgcttcatcc gcatcgacac cgcctgcgtg tgcgtgctga gccgcaaggc cgtgcgccgc 720

gcctg 725

<210> 3

<211> 716

<212> DNA

<213> pCHO1.0-beta-NGF-RRA-Fc

<400> 3

atgagcatgc tgttctacac cctgatcacc gccttcctga tcggcataca ggccgagccc 60

cacagcgaga gcaacgtgcc cgccggccac accatccccc aggcccactg gaccaagctg 120

cagcacagcc tggacaccgc cctgcgccgc gcccgcagcg cccccgccgc cgccatagcc 180

gcccgcgtgg ccggccagac ccgcaacatt accgtggacc cccgcctgtt caagaagcgc 240

cgcctgcgca gcccccgcgt gctgttcagc acccagcccc cccgcgaggc cgccgacacc 300

caggacctgg acttcgaggt gggcggcgcc gcccccttca accgcaccca ccgcagcaag 360

cgcagcagca gccaccccat tttccaccgc ggcgagttca gcgtgtgcga cagcgtgagc 420

gtgtgggtgg gcgacaagac caccgccacc gacatcaagg gcaaggaggt gatggtgctg 480

ggcgaggtga acattaacaa cagcgtgttc aagcagtact tcttcgagac caagtgccgc 540

gaccccaacc ccgtggacag cggctgccgc cgcattgaca gcaagcactg gaacagctac 600

tgcaccacca cccacacctt cgtgaaggcc ctgaccatgg acggcaagca ggccgcctgg 660

cgcttcatcc gcatcgacac cgcctgcgtg tgcgtgctga gccgcaaggc cgtgcg 716

Claims (10)

1. recombinant vector, it is characterised in that：It contains the nucleotide sequence of nerve growth factor Fc antigen-4 fusion protein genes；Described The nucleic acid sequence of nerve growth factor Fc antigen-4 fusion protein genes is：PCHO1.0-beta-NGF-Fc such as SEQ ID NO：Shown in 1, Or pCHO1.0-beta-NGF-opt-Fc such as SEQ ID NO：Shown in 2 or pCHO1.0-beta-NGF-RRA-Fc such as SEQ ID NO：Shown in 3, the carrier is Freedom^TMpCHO1.0。

2. recombinant cell, it is characterised in that：It contains recombinant vector described in claim 1；The recombinant cell is secretion table Up to the CHO-S cell strains of recombined human β-NGF-Fc fusion proteins.

3. recombinant cell according to claim 2, it is characterised in that：CHO-S cell strains are in CD Forti CHO serum-frees Routine culture in culture medium.

4. recombinant cell according to claim 2, it is characterised in that：It is by methotrexate and puromycin pressurization sieve Cell after choosing culture.

5. recombinant cell according to claim 4, it is characterised in that：The pressurization screening is sieved for the pressurization in two stages Choosing：A concentration of 0~120nM of methotrexate in first stage screening；Second stage screening in methotrexate a concentration of 50~ 1000nM；

A concentration of 0~1.2 μ g/mL of puromycin in the first stage screening；Puromycin is dense in second stage screening Degree is 0~30 μ g/mL.

6. β-NGF-Fc fusion proteins, it is characterised in that：It is by any one of the recombinant vector of claim 1 or claim 2-5 Recombinant cell be prepared.

7. the recombinant vector of claim 1 or the recombinant cell of any one of claim 2-5 are used to prepare or produce β-NGF-Fc The purposes of fusion protein.

8. the preparation method of the recombinant cell of any one of claim 2-5, it is characterised in that：Include the following steps：

(1) recombinant vector of claim 1 is transfected into Chinese hamster ovary celI, in the Tissue Culture Flask containing serum free medium Static gas wave refrigerator 10~14 days (such as 12 days), a concentration of 0~120nM of methotrexate, purine are mould in the serum free medium A concentration of 0~1.2 μ g/mL of element；

(2) cell that step (1) culture obtains is transferred in shaking flask and shakes culture 11~15 days (such as the 13rd day), every 3~4 It passes a generation, and culture medium is identical as step (1)；

(3) suitably increase cell density, continue to shake culture 18~25 days in the shaking flask containing serum free medium, every 3~4 It passes generation, a concentration of 50~1000nM, a concentration of 0~30 μ of puromycin of methotrexate in the serum free medium G/mL obtains the recombinant cell of any one of claim 2-6；

(4) optionally, further include monoclonal screening is carried out to the cell that is obtained, the mistake of cell strain to obtain higher expression quantity Journey.

9. the preparation method of β-NGF-Fc fusion proteins, it is characterised in that：Preparation method including claim 8, and culture The step of recombinant cell and harvest cells and supernatant.

10. the purification process of β-NGF-Fc fusion proteins, it is characterised in that：Include the following steps：

1) recombinant cell of culture expression β-NGF-Fc fusion proteins, harvests cells and supernatant；

2) cells and supernatant is purified with ion-exchange chromatography, obtains sample after purification；

3) sample that step 2) obtains is purified with sieve chromatography, the β-NGF-Fc fusion proteins purified；It is preferred that Ground, the recombinant cell that the recombinant cell for expressing β-NGF-Fc fusion proteins is any one of claim 2-6；Further preferably Ground, preparation method is prepared the recombinant cell according to claim 9.