CN1605592A - Protease resistant mutein of human ciliary neurotrophic factor and its preparation and use - Google Patents

Abstract

The present invention relates to one kind of human ciliary neurotrophic factor mutant with proteinase resistance, and the mutant is formed through mutating natural human ciliary neurotrophic factor. The mutation includes the mutation of site-13 amino acid residue and/or site-14 amino acid residue into any amino acid residue but arginine residue. The mutant may be he efficiency expressed in yeast expression system. Compared with available technology, the mutant of the present invention has even high bioactivity and even wide clinical application foreground, and may find its wide use in biomedicine fields.

Description

Protease resistant human ciliary nerve trophic factor mutant and production method thereof and purposes

Technical field

The present invention relates to a kind of neurotrophic factor, be specifically related to the protease resistant human ciliary nerve trophic factor mutant, also relate to the production method of this mutant in the methanol yeast cell and the purposes in field of medicaments.

Background technology

Ciliary neurotrophic factor (ciliary neurotrophic factor, be a kind of cytokine with various biological function CNTF): CNTF can promote the survival of Sensory neurone, motor neuron, cerebral neuron and hippocampal neuron; CNTF can promote the cholinergic differentiation of sympathetic neuron and make the neuroglia progenitor cell be divided into astroglia cell; CNTF can promote the ripe and survival of oligodendrocyte; Some peripheral tissues such as skeletal muscle are arranged in addition also to CNTF respond (Sleeman, M.W., et al.Pharma.Acta.Helv., 2000,74:265-272.This document and other document of being quoted in this application all as of the present invention with reference to interpretation data, just quoted as a reference separately as each piece document.)。Recombinant human CNTF (rhCNTF) once was used to treat amyotrophic lateral sclerosis (ALS), but cut and stopped clinical experiment because of side reaction reaches uncertain therapeutic efficacy too greatly.Studies show that recently CNTF can be positioned at hypothalamic acceptor and the body weight setting point (set-point) of body is regulated by activation, ingests slimming effect thereby play to reduce.For developing higher, the less adverse effect of biological activity, can be used for clinical CNTF, the researchist begins one's study and develops the CNTF mutant.AX15 is a CNTF triple mutant body by the invention of Regeneron company: 17 Cys is replaced by Ala, and 63 Gln is replaced by Arg, has lacked 15 amino acid of C-terminal.Compare with natural CNTF, AX15 has higher biologic activity, better stability and solubility.At present, AX15 has finished the III phase clinical experiment that indication is an obesity as biological products in the U.S., and the clinical experiment result by statistics credit analyse and show to have significant difference.

The carrier of yeast expression system-host system divides additional build and integrated two kinds of phraseologies.The recombinant plasmid of additional build is present in the zymic tenuigenin, have in the recombinant plasmid autonomously replicating sequence (autonomously replicating sequence, ARS).Can be in yeast cell self-replicating, and under the promoter element regulation and control expression alien gene, but this recombinant plasmid loses in going down to posterity easily in yeast host cell budding division, pedigree, thereby often is difficult to amplify production with the industrialization large fermentation tank.Integrated expression plasmid is to be incorporated into (single copy or multi-copy integration) in the zymic genome with linearization plasmid or circular plasmids form by the homologous recombination method; duplicate along with yeast host bacterium THE REPLICATION OF CHROMOSOME then; the external source goal gene goes down to posterity in yeast pedigree and tends towards stability; be difficult for losing; the process expressed with the yeast autogene of the process expressed under the regulation and control of Yeast promoter element of goal gene is the same in addition; be in nucleus, to transcribe out hnRNA; through modifying and be processed into mRNA; and pass through nuclear membrane and arrive tenuigenin and combine with rrna, express goal gene again.Integrated phraseology is fit to carry out fermentative production with the industrialization large fermentation tank.On the other hand, yeast has accumulated a large amount of experiences as the important host bacterium of industrial microorganism aspect industrial fermentation.The yeast host bacterium is a crust phase moral pichia spp, and it is a kind of industrial yeast bacterial classification, be used to since the seventies manufacture order cell protein (single cell protein, SCP).Because it can utilize methyl alcohol as the unique carbon source and the energy, thereby is called thermophilic methanol yeast (Methylotrophic yeast) or directly is called methanol yeast in industrial microorganism.Methanol yeast is used as the multiple-shaped nuohan inferior yeast (Hansenula polymorpha) and the Candida boidinii (Candida boidinii) etc. in addition of gene engineering expression system at present.Methanol yeast generally all has fermentation density height, the protein secreting ability is strong, the exogenous gene expression level is high and glycosylation modified more near advantages such as higher eucaryotes, is eukaryotic microorganisms expression system ideal in the present international biological technical field.Especially pichia pastoris phaff, several yeast of multiple-shaped nuohan inferior yeast have quite sophisticated production technique aspect the cultivation of large fermentation tank high cell density fermentation.Therefore, has advantage on many industries with lower eukaryotes yeast expression system expression alien gene: can be in substratum with the genetic engineered product direct secretion, and be that well-oxygenated environment helps disulfide linkage formation in the substratum, help giving expression to the protein molecule of native conformation.For example, nineteen eighty-two with yeast saccharomyces cerevisiae express large molecular weight protein hepatitis B surface antigen(HBsAg) (as recombinant vaccine) succeed (Valenzuela P, et al.Nature1982,298:347-350), and the approval listing.

Based on above factor, yeast expression system has become develops the of great value expression system of genetically engineered Human Ciliary Neurotrophic Factor at present in the world.Yet, when expressing AX15, find that AX15 easily is degraded with pichia pastoris phaff, therefore press for and make up a kind of protease resistant human ciliary nerve trophic factor mutant.

Summary of the invention

For a kind of protease resistant human ciliary nerve trophic factor mutant is provided, the present inventor is extensive studies through going deep into, the site that degraded takes place when expressing in the pichia pastoris phaff host cell according to AX15 has obtained the protease resistant human ciliary nerve trophic factor mutant unexpectedly.This mutant forms by the 13rd of the natural human ciliary neurotrophic factor and the 14th amino acids residue are undergone mutation individually or simultaneously.Said the 13rd and the 14th amino acids residue can sport the arbitrary amino acid residue except that arginine residues, preferably sport Methionin, leucine or alanine residue.The aminoacid sequence shown in the Sequence Identification number 1 is represented one of mutant of the present invention in the sequence table.

The present inventor has realized human ciliary nerve trophic factor mutant efficiently expressing in yeast cell by creationary research.

Mutant of recombined human ciliary nerves nutrilitc of the present invention preferably carries out scale operation with methanol yeast engineering bacteria system: a kind of engineering bacteria that is integrated the recombinant plasmid that contains the human ciliary nerve trophic factor mutant gene of a copy at least, shaking under bottle or the fermentor tank high density fermentation condition, the human ciliary nerve trophic factor mutant gene is secreted into the mutant of recombined human ciliary nerves nutrilitc expression product in the substratum under a kind of driving of methanol induction promotor.

The method of human ciliary nerve trophic factor mutant of the present invention may further comprise the steps:

(a) under the condition that is fit to the expressing human ciliary nerve trophic factor mutant, use the bioreactor culture proper host cell;

(b) from culture, isolate human ciliary nerve trophic factor mutant.

In step (a), culture density is 500-800 OD600, and every liter of dry cell weight is the 100-300 gram, and the expression amount of human ciliary nerve trophic factor mutant is greater than 100mg/L.

In step (a), expressed 4～8 days with methanol induction.

The inventor also further is optimized the element in the expression cassette, has especially increased zymic endogenous restriction enzyme site at 5 ' end, thereby expression and separation efficiency further are provided.On this basis, finished the present invention.

For ease of the molecular cloning of gene and the purpose product that makes expression after the processing of yeast entogenous proteolytic enzyme, can directly obtain the human ciliary nerve trophic factor mutant of the no Met of 5 ' end, thereby simplified follow-up separation and purifying process greatly, the present inventor has introduced restriction enzyme XhoI and yeast entogenous Proteinase K EX-2 processing site at 5 of human ciliary nerve trophic factor mutant ' end.Preferred yeast entogenous restriction enzyme site is LEKR.

Technology such as plasmid, transformed yeast cell, the screening of Yeast engineering bacteria, the fermentation of engineering bacteria and the separation of product that the human ciliary nerve trophic factor mutant gene is inserted in the method are carried out (people such as Sambrook for example by ordinary method known in the art basically, molecular cloning: experiment guide (third edition) (New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or the condition of advising according to manufacturer.Biomaterials such as used original strain and plasmid all can be by commercial acquisition.

Has an above-mentioned human ciliary nerve trophic factor mutant gene expression of gene box and an a kind of marker gene of containing in the methanol yeast expression vector of the present invention at least for screening.This recombinant expression vector transforms methanol yeast host bacterium can use cyclic plasmid form or two kinds of forms of linearizing site-specific integration carrier.The present invention preferentially selects linearizing site-specific integration carrier, and preferentially with the protoplast transformation method with in the locus in recombinant expression vector site-directed integration specificity site to methanol yeast (as the pichia pastoris phaff etc.) genome (as being incorporated into AOX1 gene or HIS4 gene locus), at least stably be integrated with the recombinant vectors that contains above-mentioned expression cassette of a copy in the engineering bacteria of production human ciliary nerve trophic factor mutant gene involved in the present invention.

The inventor finds that also the deoxynucleotide (5-11 gathers the deoxy-oligonucleotide sequence) that inserts the gene 5 ' non-coding region in yeast source before the ATG of goal gene can improve the expression amount of goal gene.The oligodeoxynucleotide that inserts is preferentially selected A and T base.The oligonucleotide sequence that is connected with human ciliary nerve trophic factor mutant gene A TG is preferentially from AOX1 and AOX2, glyceraldehyde 3-phosphate dehydro-genase gene (the glyceraldehydes-3-phosphatedehydrogenase gene of pichia pastoris phaff in the present invention, GAP), formaldehyde dehydrogenase gene (formaldehyde dehydrogenasegene, FLD1) or constructed by dihydroxy acetone synthetase (dihydroxyacetone synthase, DAS1) gene 5 ' end control region sequence.

Several regulating and controlling sequences are identified and are confirmed, can be used for expressing human ciliary nerve trophic factor mutant in pichia pastoris phaff.5 ' the control region (promoter element) of the alcohol oxidase of cloning from the pichia pastoris phaff genome (AOX1 and AOX2) gene, glyceraldehyde 3-phosphate dehydro-genase gene (GAP), formaldehyde dehydrogenase gene (FLD1), constructed by dihydroxy acetone synthetase (DAS1) gene and p40 gene can be used for driving the human ciliary nerve trophic factor mutant genetic expression in downstream.Preferential 5 ' the control region selected of the present invention is for example selected AOX1, (Stroman DW, et al.US4,855,231) such as AOX2, GAP, FLD1 and DAS1.5 ' control region that override of the present invention is selected is to select the AOX1 promoter sequence.

The function of 3 ' terminator sequence is that the structure gene of encoding is played termination, poly-adenosine and stable mRNA.Any this area 3 ' terminator sequence commonly used all can be used for the present invention, for example derives from 3 ' terminator sequence of inferior multiform yeast of the Chinese and pichia pastoris phaff.Preferential 3 ' the terminator sequence that adopts those from pichia pastoris phaff bacterium source, for example 3 ' terminator sequence of AOX1 gene, AOX2 gene, DAS1 gene, p40 gene and His4 gene is especially preferentially selected AOX13 ' terminator sequence.

In the present invention, the human ciliary nerve trophic factor mutant gene clone that builds is in a kind of suitable pichia pastoris phaff secretion expression and the born of the same parents on the expression vector, as pPIC9, pPIC9K, pPIC3, pPIC3K, pAO804, pAO815, pHIL-S1, pHIL-D2, pHWO10, pPICZ, pPICZ α, pGAPZ, pGAPZ α, pYAM7SP6 etc.The present invention preferentially selects to use pPIC9 and pPIC9K secretion expression plasmid as expression vector.

The recombinant plasmid that above-mentioned structure is finished directly or be transformed into pichia pastoris phaff host mycetocyte after linearizing, wherein more sophisticated transformation technology has, as with the chemical conversion process of lithium chloride (Ito, et al.JBacteriol.1983,153:163-168); Electroporation method for transformation (Bio-Rad laboratories 1991, Genepulser transfection apparatus operating instruction and applications guide.); The protoplast transformation method (Cregg JM, et al.Mol.Cell Biol.1985,5:3376-3385).The preferential protoplast transformation method of selecting people such as Cregg to provide in scientific research of the present invention, recombinant plasmid linearizing and identify that recombinant vectors is integrated and use the Southern hybridization technique.

It is methanol yeast that the present invention is used for as genetically engineered host bacterium.Methanol yeast comprises candiyeast (Candida), debaryomyces hansenii (Hansenula), pichia spp (Pichia) and four genus of torulopsis (Torulopsis), and wherein all methanol yeast bacterial strains all can be used as the host bacterium of inventor's ciliary nerve trophic factor mutant expression of gene.Preferentially select pichia pastoris phaff and multiple-shaped nuohan inferior yeast in the present invention, especially preferentially select auxotroph pichia pastoris phaff bacterial strain GS115 (NRRLY-15851), GS190 (NRRLY-18014), PPFI (NRRLY-18017), KM71 and protease-deficient pichia pastoris phaff bacterial strain SMD1168 (pep4 Δ his4 (Mut+his-)), the Microbiological Characteristics of these bacterial strains is described in detail, and (U.S.patent 4,818,700 and U.S.patent 4,812,405).The inventor also finds, auxotroph pichia pastoris phaff bacterial strain is convenient to the effect of screening, recombinating and transforming in addition.

Wild-type pichia pastoris phaff bacterial strain such as NRRLY-11430 and NRRLY-11431 also can be directly as the host bacterium of inventor's ciliary nerve trophic factor mutant gene, as long as one of clone can be for the marker gene (as SUC2 gene and neo gene etc.) of screening on expression vector, after recombinant vectors transforms the wild-type Pichi strain like this, wherein transformant just can or contain on the antibiotic substratum of G418 at the substratum that contains sucrose and grows, but not transformant then can not grow on this type of substratum, so just can filter out recombinant conversion easily.

In preference of the present invention, replace the signal peptide sequence (signal peptide sequence in also available other yeast host bacterium sources and the chimeric signal peptide sequence of synthetic) of yeast saccharomyces cerevisiae α mating gene with alcohol oxidase (AOX1 and AOX2) gene preference codon, insert 5 poly-oligodeoxynucleotides (AAACGATG) in human ciliary nerve trophic factor mutant gene A TG front, wherein contain eukaryotic gene conservative kozak sequence A XXATGG (Kozak M.Nucleic Acids Res.1987,15:8 125-8148; KozakM.Proc.Natl.Acad.Sci.USA 1990,87:8301-8305); Insert the gene order in yeast saccharomyces cerevisiae KEX-2 proteolytic enzyme processing site (Leu-Glu-Lys-Arg) at gene 5 ' end, (alpha-mating factor, α-MF) Prepro sequence (85 the amino acid whose gene orders of encoding) constitutes mosaic gene to the α-mating factor in goal gene and yeast saccharomyces cerevisiae source.These two kinds of gene fragments are connected with the AOX1 gene 5 ' control region (pichia pastoris AOX1 5 ' regulation region) of upstream pichia pastoris phaff again, contain promoter element and enhancer element at the AOX1 control region.

In another preference, the present invention will be suitable for human ciliary nerve trophic factor mutant gene clone that pichia spp host bacterium expresses to the AOX1 5 ' control region of pPIC9 expression vector etc. and the centre of AOX1 3 ' terminator sequence, proofread and correct reading frame, on expression vector, constitute an expression cassette (expressioncassette), expression vector (can be cyclic or linearizing) with this gene recombination passes through the protoplast transformation method again, transform the Pichia yeast protoplastis, and then expression cassette stably is incorporated in the genome of host bacterium (being on the karyomit(e)).This expression cassette AOX1 5 ' control region or promotor are used to strengthen the speed that human ciliary nerve trophic factor mutant and mutant mRNA transcribe; AOX1 3 ' terminator sequence has function and the effect (as impelling hnRNA to add polyA tail etc.) that stops human ciliary nerve trophic factor mutant gene translation and stable mRNA transcription product.

In the present invention, for large-scale industrial production gene recombination human ciliary nerve trophic factor mutant product, according to methanol yeast, especially the microbiological property of pichia pastoris phaff-integrated expression system, on fermentor tank, formulated for three stages, the schedule of operation of the fermentation engineering of high-density and batch feeding (fermentation protocol): 1. fs, be also referred to as vegetative period, the engineering bacteria of the highest level expressing human ciliary nerve trophic factor mutant that above-mentioned screening and separating is obtained connects bacterium at a primary yeast basic medium (10 X Basal Salts+250 X PTM as kind of a daughter bacteria ₁+ 5% glycerine) in, the carbon source in this substratum is a kind of non-induction type carbon source (as glycerine).When growing in the substratum that contains this carbon source, this methanol yeast engineering bacterium expression foreign gene is suppressed fully, promptly only allows cell fission, propagation, and cell density increases, and foreign gene is not expressed, and maintains 3-6 at the pH of growth phase substratum.2. subordinate phase, this is of short duration period, non-induction type carbon source consumes gradually makes a gift of most induction type carbon source (methyl alcohol) and begins to mend fermentor tank lentamente, cell density in the fermentor tank is in further increase, and the holddown of methyl alcohol reaction promotor (methanolresponsive promoter) is progressively being removed.Progressively adjusted to the pH value of production phase at the pH of this stage substratum, promptly generally in the pH3-6 scope.3. the phase III, also claim the production phase, begin to accelerate to mend in the fermentor tank that what the cell density in the fermentor tank had just no longer increases (as mut at this stage induction type carbon source methyl alcohol ^-What bacterial strain), have also has increase slightly (as mut ⁺Bacterial strain), the human ciliary nerve trophic factor mutant gene begins gene expression product to secrete continuously in the fermention medium under methyl alcohol reacts the regulation and control of promotor (as the AOX1 promotor), this fermentation mode is called restriction methyl alcohol batch feeding mode (limited methanol fed-batch mode), and the methanol concentration in whole production stage fermentation jar is controlled at it in concentration range of 0.5-0.9% by the online detection of methyl alcohol electrode.Fermentative production human ciliary nerve trophic factor mutant of the present invention, in the phase III, it is the production phase, also can adopt the feed supplement mode (mixed feed fed-batch mode) of mixing, be to mend in the fermentor tank gradually after non-induction type carbon source glycerine adds (2: the 1) mixing by a certain percentage of induction type carbon source methyl alcohol, the characteristics of this fermentation mode are, cell density in the fermentor tank the production phase also in further increase, contain human ciliary nerve trophic factor mutant expression of gene box and under induction type carbon source methanol induction, human ciliary nerve trophic factor mutant be secreted in the fermention medium.Mix the feed supplement mode and especially be fit to do to continuously ferment, continue to produce goal gene product human ciliary nerve trophic factor mutant with pichia pastoris phaff host bacterium.

In the present invention, the human ciliary nerve trophic factor mutant product obtains the secretion expression in pichia pastoris phaff, the expression product direct secretion is in substratum, fermented liquid internal secretion expression levels is up to 100mg/L, by the optimization of fermentation manufacturing technique, Yeast engineering bacteria also has the potentiality that further improve expression amount.

For realizing efficiently expressing, the present invention also provides a kind of expression vector, and this expression vector contains the dna sequence dna of the above-mentioned human ciliary nerve trophic factor mutant of code book invention.

The present invention also provides a kind of host cell, and it contains the dna sequence dna of above-mentioned expression vector of the present invention or above-mentioned coding human ciliary nerve trophic factor mutant.This host cell is the methanol yeast cell, preferred pichia pastoris phaff.In its genome, be integrated with the dna sequence dna of coding human ciliary nerve trophic factor mutant, and operationally link to each other at the 5 ' control region in 5 of the dna sequence dna of described coding human ciliary nerve trophic factor mutant ' end upstream and pichia pastoris phaff bacterium source, described 5 ' control region comprises and includes promoter element and Kozak sequence enhancer element;

3 of the dna sequence dna of described coding human ciliary nerve trophic factor mutant ' end downstream operationally links to each other with the 3 ' terminator sequence in methanol yeast source.

Described 5 ' control region is selected from 5 ' control region of following gene: alcohol oxidase (AOX1 and AOX2) gene, glyceraldehyde 3-phosphate dehydro-genase gene (GAP), formaldehyde dehydrogenase gene (FLD1), constructed by dihydroxy acetone synthetase (DAS1) gene or histidinol deoxygenase (HIS4) gene in pichia spp source;

Described 3 ' terminator sequence is selected from 3 ' terminator sequence of following gene: AOX1 gene, AOX2 gene, p40 gene and the His4 gene in pichia pastoris phaff source.

Between the dna sequence dna and 5 ' control region of described coding human ciliary nerve trophic factor mutant, also contain the encoding sequence of the α-mating factor gene leader peptide sequence in yeast saccharomyces cerevisiae source.

Described Kozak sequence enhancer element is AAACGATG.

Described host cell comprises Mut-and Mut+ phenotype.

Described host cell is included in and is integrated with the present invention's bacterial strains such as the GS115 of dna sequence dna of human ciliary nerve trophic factor mutant and SMD1168 of encoding in its karyomit(e).

Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Compared with prior art, protease resistant human ciliary nerve trophic factor mutant of the present invention has higher biological activity and potential applicability in clinical practice better.

Description of drawings

Fig. 1 is the structure collection of illustrative plates of a kind of expression plasmid of yeast pPIC9.

Fig. 2 is the electrophorogram of AX15 expression product in pichia pastoris phaff.

Fig. 3 is determining of AX15 degradation site.

Fig. 4 is the structure of AX15 (R14K) gene.

Fig. 5 is the electrophorogram of AX15 (R14K) expression product in pichia pastoris phaff.

Fig. 6 is the purge process of AX15 (R14K).

Fig. 7 is the determination of activity of AX15 (R14K).

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are interpreted as only being used to the present invention is described and are not used in restriction the present invention.The experimental technique of unreceipted actual conditions is routine or art methods in the following example.

One, material and method

(1) bacterial classification:

1. pichia pastoris phaff host bacterium GS115 his4 (Mut+his-) NRRLY-15851; KM71aox1 Δ ∷ SARG4his4arg4 (Mut ^sHis ^-); SMD1 168 pep4 Δ his4 (Mut+his-).

2. e. coli host bacteria:

E.coli?Top10?F′{proAB，lacI ^q，lacZΔM15，Tn10(Tet ^R)}mcrA，Δ(mrr-hsdRMS-mcrBC)，φ80lacZΔM15，ΔLacX74，deoR，recA1，araD139，Δ(ara-leu)7697，galU，galK，epsL(str ^R)，endA?1，nupGλ ^-；

E.coli?JM109?F′[(recA1，supE44?endA1?hsdR17?gyrA96?relA1?thiΔ(lac-proAB)F′[traD36?proAB ⁺lacI ^q?lacZ?ΔM15])；

E.coli?HB101(supE44?hsd?S20(rB-mB-)recA ₁₃ara-14?proA ₂?lac?Y ₁?galK ₂rpsL ₂₀?xyl-5?mtl-1)。

Above bacterial strain is all by commercial acquisition.

(2) main agents

1.DNA restriction enzyme, T ₄Dna ligase, polysaccharase etc. are respectively available from GIBCO-BRL, Pharmacia, Bio-Labs and magnificent biotechnology company limited

2. casein hydrolysate (German MERK company product)

3.Bacto-yeast extract (U.S. Difco company product)

4.PCR amplification kit (Sweden Pharmacia company product)

5.DNA sequential analysis test kit (U.S. USB company product)

6. isotropic substance α-P ³²-dATP and γ-P ³²-dATP (Britain Amersham company product)

7. some solution preparations of yeast protoplast transformation method

(1) SED:1M sorbyl alcohol, 25mM EDTA, 50mM DTT, pH8.0

(2) SCE:9.1g sorbyl alcohol, 1.47g Trisodium Citrate, 0.168gEDTA, pH5.8

(3) CaS:1M sorbyl alcohol, 10mM CaCl ₂

(4) SOS:1M sorbyl alcohol, 0.3 X YPD, 10mM CaCl ₂

(5)CaT：20mM?Tris-HCl，pH7.5，20mM?CaCl ₂

(6)PEG：20％PEG-3350，10mM?CaCl ₂，10mM?Tris-HCl(pH7.4)

(7)1M?PBS?buffer：132ml?1M?K ₂HPO ₄，868ml?1M?KH ₂PO ₄，pH6.0。

Two, the expression of AX15 gene in pichia pastoris phaff

Expression vector pPIC9-AX15 is transformed GS115 with the protoplast transformation method, 24 clones of picking at random from the RDB culture plate, be inoculated in the 4ml BMMY nutrient solution, 30 ℃ of shaking table joltings 36-48 hour, the centrifugal 10min of 5000rpm respectively got 100 μ L supernatant liquors, vacuum is drained, sample is carried out the 15%SDS-PAGE electrophoresis respectively to be identified, compares with the unloaded vector expression thing of pPIC9, identifies high-expression clone.The high-expression clone that these are screened is protected bacterium, slant culture with 15% glycerine low temperature respectively then, is used for as the engineering bacteria of further expressing or does Southern hybridization and methyl alcohol utilizes the usefulness of evaluation such as phenotype.

The colony inoculation of the high-expression clone of the above-mentioned through engineering approaches of picking in 4ml BMGY liquid nutrient medium, 30 ℃ of shaking table shaken overnight.Getting the 1ml yeast liquid transfers in 50ml BMGY nutrient solution, continue jolting 18-24 hour, connecing bacterium amount by 4% again connects 40ml kind daughter bacteria and goes in the 1000ml BMGY substratum, continue vibration 24-30 hour at 30 ℃ of shaking tables, nutrient solution with the centrifugal 5min of 5000rpm, is abandoned supernatant, and these two kinds of engineering bacterias are used 200ml BMMY abduction delivering substratum suspension thalline respectively then, be placed on 30 ℃ of shaking tables and continue vibration 3-4 days, added (adding to final concentration with 100% methyl alcohol is 0.5%) every 24 hours.At this moment the cell density of pichia pastoris phaff engineering bacteria has generally reached 18-20 OD ₆₀₀, the foreign protein great majority of genetic expression are secreted in the liquid nutrient medium.Fermention medium is abandoned thalline at 4 ℃ of centrifugal 20min of 10000rpm, contains the supernatant liquor of a large amount of AX15 expression products, and sample can be used for the SDS-PAGE electrophoresis to be identified, sees Fig. 2.

Three, the AX15 degradation site determines

AX15 and degraded product thereof are carried out N-terminal sequence mensuration respectively, the results are shown in Figure 3.N-terminal sequence analytical results shows that the bigger band of molecular weight is complete AX15 molecule, and the less band of molecular weight is to have lacked 13 amino acid whose degraded products of N-end.

Four, the structure of AX15 (R14K) gene

Use primer 1:5 ' CC CTC GAG AAA AGA GCT TTC ACA GAG CAT TCA CCGCTG ACC CCT CAC CGT AAG GAC 3 ' and primer 2: 5 ' GG GAA TTC CTA TTACCC AGT CTG ATG AGA AGA AAT 3 ' is that template amplification goes out AX15 (R14K) gene with pPIC9-AX15, sees Fig. 4.

Five, the expression of AX15 (R14K) gene in pichia pastoris phaff

Expression vector pPIC9-AX15 (R14K) is transformed GS115 with the protoplast transformation method, 24 clones of picking at random from the RDB culture plate, be inoculated in the 4ml GMMY nutrient solution, 30 ℃ of shaking table joltings 36-48 hour, the centrifugal 10min of 5000rpm respectively got 100 μ L supernatant liquors, vacuum is drained, sample is carried out the 15%SDS-PAGE electrophoresis respectively to be identified, compares with the unloaded vector expression thing of pPIC9, identifies high-expression clone.The high-expression clone that these are screened is protected bacterium, slant culture with 15% glycerine low temperature respectively then, is used for as the engineering bacteria of further expressing or does Southern hybridization and methyl alcohol utilizes the usefulness of evaluation such as phenotype.

The colony inoculation of the high-expression clone of the above-mentioned through engineering approaches of picking in 4ml BMGY liquid nutrient medium, 30 ℃ of shaking table shaken overnight.Getting the 1ml yeast liquid transfers in 50ml BMGY nutrient solution, continue jolting 18-24 hour, connecing bacterium amount by 4% again connects 40ml kind daughter bacteria and goes in the 1000ml BMGY substratum, continue vibration 24-30 hour at 30 ℃ of shaking tables, nutrient solution with the centrifugal 5min of 5000rpm, is abandoned supernatant, and these two kinds of engineering bacterias are used 200ml BMMY abduction delivering substratum suspension thalline respectively then, be placed on 30 ℃ of shaking tables and continue vibration 3-4 days, added (adding to final concentration with 100% methyl alcohol is 0.5%) every 24 hours.At this moment the cell density of pichia pastoris phaff engineering bacteria has generally reached 18-20 OD ₆₀₀, the foreign protein great majority of genetic expression are secreted in the liquid nutrient medium.Fermention medium is abandoned thalline at 4 ℃ of centrifugal 20min of 10000rpm, contains the supernatant liquor of a large amount of AX15 (R14K) expression product, and sample can be used for the SDS-PAGE electrophoresis to be identified, sees Fig. 5.

Six, purifying and the determination of activity of AX15 (R14K)

And then culture supernatant has been carried out the research of pilot scale purifying process aspect: nutrient solution carries out centrifugal 5000g * 20min earlier, abandons thalline, and supernatant liquor is through ultrafiltration and concentration, and behind the gel-filtration purifying, the purity of AX15 (R14K) is greater than 90%.The SDS-PAGE of sample the results are shown in Figure 6 after each purification procedures.The experiment of TF-1 cell survival shows the EC of AX15 (R14K) ₅₀Be 200ng/mL (Fig. 7), be 4-5 times of rhCNTF, suitable with AX15.

Sequence table

＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A

＜120〉protease resistant human ciliary nerve trophic factor mutant and production method thereof and purposes

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<170>PatentIn?version?3.2

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<400>1

Met?Ala?Phe?Thr?Glu?His?Ser?Pro?Leu?Thr?Pro?His?Arg?Lys?Asp?Leu

1 5 10 15

Ala?Ser?Arg?Ser?Ile?Trp?Leu?Ala?Arg?Lys?Ile?Arg?Ser?Asp?Leu?Thr

20 25 30

Ala?Leu?Thr?Glu?Ser?Try?Val?Lys?His?Gln?Gly?Leu?Asn?Lys?Asn?Ile

35 40 45

Asn?Leu?Glu?Ser?Ala?Glu?Gly?Met?Pro?Val?Ala?Ser?Thr?Asp?Arg?Trp

50 55 60

Ser?Glu?Leu?Thr?Glu?Ala?Glu?Arg?Leu?Gln?Asp?Asn?Leu?Gln?Ala?Try

65 70 75 80

Arg?Thr?Phe?His?Val?Leu?Leu?Ala?Arg?Leu?Leu?Glu?Asp?Gln?Gln?Val

85 90 95

His?Phe?Thr?Pro?Thr?Glu?Gly?Asp?Phe?His?Gln?Ala?Ile?His?Thr?Leu

100 105 110

Leu?Leu?Gln?Val?Ala?Ala?Phe?Ala?Try?Gln?Ile?Glu?Glu?Leu?Met?Ile

115 120 125

Leu?Leu?Glu?Try?Lys?Ile?Pro?Arg?Asn?Glu?Ala?Asp?Gly?Met?Pro?Ile

130 135 140

Asn?Val?Gly?Asp?Gly?Gly?Leu?Phe?Glu?Lys?Lys?Leu?Trp?Gly?Leu?Lys

145 150 155 160

Val?Leu?Gln?Glu?Leu?Ser?Gln?Trp?Thr?Val?Arg?Ser?Ile?His?Asp?Leu

165 170 175

Arg?Phe?Ile?Ser?Ser?His?Gln?Thr?Gly

180 185

Claims (16)

1. protease resistant human ciliary nerve trophic factor mutant, it is characterized in that this mutant is to be formed by natural human ciliary neurotrophic factor sudden change, said sudden change comprises that its 13rd and the 14th amino acids residue sport the arbitrary amino acid residue except that arginine residues individually or simultaneously.

2. the described protease resistant human ciliary nerve trophic factor mutant of claim 1 is characterized in that the said the 13rd and the 14th amino acids residue is Methionin, leucine or alanine residue.

3. the described protease resistant human ciliary nerve trophic factor mutant of claim 1 is characterized in that this mutant has the aminoacid sequence shown in the Sequence Identification in the sequence table number 1.

4. expression vector is characterized in that it contains the dna sequence dna of the described aminoacid sequence of coding claim 3.

5. host cell is characterized in that it contains the described expression vector of claim 4.

6. the described host cell of claim 5 is characterized in that this host cell is the methanol yeast cell.

7. the described host cell of claim 6 is characterized in that said methanol yeast cell is a pichia pastoris phaff.

8. the described host cell of claim 7, it is characterized in that being integrated with in this host cell gene group the dna sequence dna of coding human ciliary nerve trophic factor mutant, and operationally link to each other at the 5 ' control region in 5 of the dna sequence dna of said coding human ciliary nerve trophic factor mutant ' end upstream and pichia pastoris phaff bacterium source, said 5 ' control region comprises and includes promoter element and Kozak sequence enhancer element, and 3 of the dna sequence dna of said coding human ciliary nerve trophic factor mutant ' end downstream operationally links to each other with the 3 ' terminator sequence in methanol yeast source.

9. the described host cell of claim 8 is characterized in that said 5 ' control region is selected from 5 ' control region of following gene: alcohol oxidase (AOX1 and AOX2) gene, glyceraldehyde 3-phosphate dehydro-genase gene (GAP), formaldehyde dehydrogenase gene (FLD1), constructed by dihydroxy acetone synthetase (DAS1) gene or histidinol deoxygenase (HIS4) gene in pichia spp source; Said 3 ' terminator sequence is selected from 3 ' terminator sequence of following gene: AOX1 gene, AOX2 gene, p40 gene and the His4 gene in pichia pastoris phaff source.

10. the described host cell of claim 9 is characterized in that between the dna sequence dna and 5 ' control region of said coding human ciliary nerve trophic factor mutant, also contains the encoding sequence of the α-mating factor gene leader peptide sequence in yeast saccharomyces cerevisiae source.

11. claim 8,9,10 described host cells is characterized in that said Kozak sequence enhancer element is AAACGATG.

12. the described host cell of claim 11 is characterized in that said host cell comprises Mut-and Mut+ phenotype.

13. a method of producing claim 1,2 or 3 described human ciliary nerve trophic factor mutant is characterized in that it may further comprise the steps:

(a) under the condition that is fit to the expressing human ciliary nerve trophic factor mutant, with the said host cell of bioreactor culture claim 5;

(b) from culture, isolate human ciliary nerve trophic factor mutant.

14. the described method of claim 13 is characterized in that in step (a) culture density is 500-800 OD600, every liter of dry cell weight is the 100-300 gram, and the expression amount of human ciliary nerve trophic factor mutant is greater than 100mg/L.

15. the described method of claim 13 is characterized in that, in step (a), expresses 4～8 days with methanol induction.

16. claim 1, the purposes of 2 or 3 described protease resistant human ciliary nerve trophic factor mutant in biological medicine.

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