CN1403481A - Production process of neurotrophic factor - Google Patents

Abstract

The present invention discloses the production process of neurotrophic factor. The production process includes computer-aided design, modifying and altering the gene of ciliary nerve trophic factor to obtain modified mutation human ciliary nerve trophic factor cDNA, artificially synthesizing the cDNA, constituting recombinant plasmid, converting to bacillus coli, high density expression in automatic fermentation tank; renaturation in tangent flow superfiltering mode; ion exchange chromatography, hydrophobic chromatography and molecular sieve chromatography to purify and thus to obtain high-purity ciliary nerve trophic factor. The present invention has the advantages of stable and efficient expression of mutant in bacillus coli, less polarization insuperfiltering course and high purity of renaturated ciliary nerve trophic factor.

Description

The production method of neurotrophic factor

Technical field

The present invention relates to the genetically engineered field, relate in particular to the production method of neurotrophic factor.

Background technology

CNTF be the earliest external discovery have the survival that promotes chicken embryo ciliary ganglion and found (Manthorpe et al., 1980, J.Neurochem.34:69-75), in decades afterwards, have many relevant biological functions to be found again.Discoveries such as Hughes it can promote perinatal period the mouse vision and cranial nerve in neuroglia progenitor cell differentiation (Hughes et al., 1988, Nature 335:70-73), but also discovery can promote growth (the Skaper and Varon of chick embryonic dorsal root ganglion, 1986, Brain Res.389:39-46).In addition, he can also promote growth and differentiation (Ip, et al.1991, the J.Neurosci.11:3124-3134 of motor neuron, hippocampal neuron and parasympathetic spinal neuron; Blottner, et al.1989, Neurosci.Lett.105:316-320).

In view of the various biological function of rhCNTF, so he is considered to can play a role in repairing nerve damage, treatment neuratorphy disease, therefore has good clinical application prospect.Finding also in the research that further CNTF particularly also can work in the obesity of Leptin tolerance in treatment of obesity, showing possible clinical application (Isabelle Gloaguen, et al.1997, institute of NAS newspaper).Therefore, a kind of method of can high reactivity, producing rhCNTF cheaply of exploitation will have Practical significance.CNTF was once cloned and expressed (Masiakowski, et al.1991, J.Neurosci.57:1003-1012 by bacterial system; Annalise Di Marco et al.1996, institute of NAS newspaper), carry out scientific research but these methods all only are applicable to the preparation small amount of sample, and be not suitable for mass preparation.

Summary of the invention

The objective of the invention is to propose a kind of production method of neurotrophic factor of easy, quick, the high reactivity rate of recovery that is suitable for suitability for industrialized production

The production method of neurotrophic factor is to pass through computer aided design (CAD), gene to Human Ciliary Neurotrophic Factor is modified transformation, obtain to transform the cDNA of back sudden change Ciliary Neurotrophic Factor Gene, this cDNA of synthetic, construction recombination plasmid, transformed into escherichia coli is expressed through the automatic fermenter high-density; Carry out renaturation in the cross-flow ultrafiltration mode; With ion exchange chromatography, hydrophobic chromatography and sieve chromatography sequential combination purifying, produce and obtain highly purified ciliary neurotrophic factor.

Advantage of the present invention:

1) adopts computer aided design (CAD), natural codon is optimized, realized the stable high expression level of mutant in intestinal bacteria.

2) adopt cross-flow ultrafiltration mode renaturation, can reduce the polarization phenomena in the ultra-filtration process, renaturation is in the may command denaturing agent and removes on the process of speed, shorten the renaturation time, make the renaturation yield up to 70%, disposable multiple scale can reach 1-10g, and used ultrafiltration material is convenient to NaOH and is carried out original position cleaning and sterilization, can adapt to the GMP requirement of pharmaceutical protein Development and Production.Adopt this renaturation mode simultaneously, also can after renaturation be finished, directly adjust the difference in flow of pump, and the large volume protein solution is concentrated so that the convenience of subsequent purification technological operation with save time.

3) ion exchange chromatography, three kinds of different separating mechanism chromatography sequential combination of hydrophobic chromatography and sieve chromatography, make the CNTF after the renaturation be able to purification refine, detect through RPHPLC (reversed-phase high-performance liquid chromatography) (HPLC-RP) and capillary electrophoresis (CE), its purity is all greater than 95%.

Description of drawings

Fig. 1 is the sudden change CNTF cDNA sequence chart of synthetic;

Fig. 2 is a CNTF recombinant plasmid structure iron;

Fig. 3 is a renaturation device synoptic diagram, among the figure: 1 basin, 2 renaturation reactors, 3 agitators, 4 ultra-filtration membrane bags, 5 peristaltic pumps, 6 waste liquid cylinders.

Embodiment

In order to improve proteic stability of CNTF and biologic activity, CNTF compares with natural human, and primary structure is changed, and it is characterized in that: the 17th halfcystine becomes L-Ala (Cys ₁₇→ Ala ₁₇), the 63rd L-glutamic acid becomes arginine (Gln ₆₃→ Arg ₆₃), and remove terminal 15 amino acid.At the e. coli codon preference characteristic different, the CNTF gene order is optimized design [seeing Fig. 1] simultaneously with the people.Particularly transform the pairing codon of each amino acid in the CNTF natural gene sequence as codon that intestinal bacteria are had a preference for exactly, consider the G+C/A+T ratio balance of whole gene order simultaneously, and at gene 5 ' end, 3 ' end and middle introduce specific restriction enzyme site respectively, as XhoI, EcoRI, AflIII is with convenient follow-up clone, splicing.Based on the swing theory of codon, the design of the CNTF gene order of our indication can also have many changes to extend.And be not limited only to the listed sequence of figure one.Then, according to the needs of gene fragment synthetic,, externally be spliced into complete sequence with the CNTF gene salvage of design.The CNTF sequence that splicing is good, order-checking correct back amplification obtains more CNTF cDNA, and be implemented in the expression vector pGENS[that relies on t7 rna polymerase and see figure two] become expression type recombinant chou pGENS CNTF, Transformed E .coli BL21 (DE3) plysS, the screening back obtains to express the positive colony of CNTF, obtains containing the inclusion body of target protein after fermentation, centrifugal collection, homogenate fragmentation.

Above clone, conversion, screening operation are conventional Protocols in Molecular Biology all, specifically see also works such as " MolecularCloning " J.Sambrook; Work such as " Short Protocols in Molecular Biology " FrederickM.Ausubel, and other molecular biology experiment technical manual.

According to Fig. 3, form the renaturation device, adopting molecular weight cut-off is ultra-filtration membrane bag or the external-compression type hollow fiber column of 3000-10000, makes up in the tangential flow mode.Wherein the material ultra-filtration membrane is acetyl cellulose, cellulose nitrate ester, inclined to one side tetrafluoroethylene or polysulfones.

To treat that recombinant protein and initial renaturation solution place the renaturation reactor, extract protein liquid out with peristaltic pump, the ultra-filtration membrane functional layer of flowing through side, denaturing agent sees through ultra-filtration membrane and injects the waste liquid cylinder because of Ultrafiltration, albumen then answers its molecular weight bigger, can't pass ultra-filtration membrane, stress newly to get back in the renaturation reactor along membranous wall, to store simultaneously liquid at the same rate and inject the renaturation reactor, it is constant that the reactor internal volume is kept, and through constantly circulation, denaturing agent concentration progressively descends in the reactor, metaprotein is finished the dehydration and the folding process on hydration surface under the condition of gentleness, until renaturation.

Refolded protein still contains foreign protein, thermal source, nucleic acid, must further remove, and the present invention has designed ion-exchange, chromatography connects hydrophobic chromatography, after connect the sequential combination of sieve chromatography.The ion exchange chromatography filler is DEAE, Q or QAE anionite.The CNTF iso-electric point is between 5.8-6.2, and pH is suitable for 7.0-9.0, can select 20-50mM imidazole hydrochloride, veronal hydrochloric acid, Tris hydrochloric acid, borax-boric acid or phosphoric acid buffer for use, and the scope of pH is moving phase at 7.0-9.0.Diluent is as last sample balance liquid in the time of also can be directly with renaturation, the counterion exchange column, thereafter renaturation is concentrated good CNTF protein solution and directly go up sample, and carry out wash-out with the above-mentioned corresponding addition 0.01-0.5NaCl gradient that flows, pass through ion exchange chromatography, the CNTF albumen of the false folding that renaturation process can be produced and part iso-electric point and the inconsistent foreign protein of CNTF are removed, and simultaneously, also can remove the host DNA fragment of quite a few.Drainage column is selected butyl-for use, and the dewatering filling of phenyl-or octyl base can be selected 20-50mM Tris hydrochloric acid, imidazoles-hydrochloride buffer or phosphoric acid buffer for use, and the scope of pH adds 0.4-1.6mol/L (NH at 6.0-9.0 ₄) ₂SO ₄Or 0.4-1.8mol/L Na ₂SO ₄Be mobile phase A, not contain (NH ₄) ₂SO ₄Or Na ₂SO ₄Damping fluid be Mobile phase B.The target peak that ion-exchange is collected is directly added 0.4-1.6mol/L (NH ₄) ₂SO ₄Or 0.4-1.8mol/L Na ₂SO ₄, can be splined on drainage column, add 0.1-1.0N (NH with above-mentioned moving phase then ₄) ₂SO ₄Or Na ₂SO ₄The balance pillar washes nucleic acid and the abundant wash-out of pyrogen quilt with initial damping fluid behind the end of the sample, thereafter with 20-50mM, and pH7.0-9.0, Tris hydrochloride buffer or imidazole buffer and water is stepwise elution sequentially.The CNTF target peak is in the elution peak of Tris hydrochloric acid or imidazoles-hydrochloride buffer.The target peak of collecting behind hydrophobic chromatography, can directly be splined on 5-50mM phosphoric acid salt is good Sephacryl of moving phase balance or Superdex molecular sieve chromatography, makes with extra care.

The used this method of the present invention is intended to the oligomer that may exist and protein degradation when further removing, system under the aforementioned hydrophobic conditions can be changed and be 5-50mM phosphoric acid salt, the target protein CNTF that obtains through this sieve chromatography, can be directly used in the preparation of preparation, and need not adopt dialysis to wait other processing that changes solution system, reduce possible pollution and loss in the treatment step.

Embodiment C NTF engineering bacteria makes up by gene Synesis Company is synthetic and clones on the T--carrier and after sequence verification, purified recovery, and with NdeI, the BamHI enzyme is cut back repurity and is reclaimed; The pGENS plasmid is equally with NdeI, BamHI XhoI, and enzyme is cut degraded, reclaims big fragment; The CNTF gene fragment that reclaims is connected outward with the large stretch of segment body of carrier, constitutes recombinant plasmid pGENS CNTF, see Fig. 2.Recombinant plasmid transformed intestinal bacteria recipient bacterium BL21 (DE3) pLYSS, through enzyme cut, proof procedure such as PCR, dna sequencing, therefrom screen a right-on transformant of structure.Through shaking a bottle expression screening, determine that pGENS CNTF2 uses original engineering bacteria for producing.

PGENS CNTF plasmid is the expressive plasmid that our company makes up voluntarily, comprises the ColE1 replicon of following element: E.coli; The Ampicillin resistant gene.

Inclusion body solubilising renaturation

Connect the renaturation device by Fig. 3.Inclusion body solubilising liquid is mixed in the initial liquid of renaturation with certain proportion.The initial liquid formula of renaturation:

Urea: 3-6MTris-HCl:5-50mM, pH6.5-8.5EDTA:5Mm recombinant protein concentration: 0.2-1.0mg/ml stores liquid formula: Tris-HCl:5-50mM, pH6.5-8.5EDTA:5mM

The volume ratio of storage liquid and renaturation solution is controlled at 1: 3-1: 10, and the renaturation reactor places on the magnetic stirring apparatus, evenly stirs.Open peristaltic pump and extract protein liquid out, the ultra-filtration membrane functional layer of flowing through side, denaturing agent sees through ultra-filtration membrane and injects the waste liquid cylinder because of Ultrafiltration, stores liquid simultaneously and also is stirred immediately by equal-volume suction renaturation reactor.In this process, can regulate the speed that the adjustable joint denaturing agent of pumping capacity and exit end pipeline bore is removed.In this working cycle, denaturing agent concentration progressively descends, and albumen then answers its molecular weight bigger, can't pass ultra-filtration membrane, stresses newly to get back to along membranous wall and carries out renaturation in the renaturation reactor.Whole renaturation process needs 5-10 hour approximately.

The preparation of CNTF purifying

With 20mM pH8.0Tris-hydrochloric acid is initial moving phase, 5 times of column volume balance Q-Sepharose FF ion exchange columns, thereafter renaturation is concentrated good CNTF protein solution and directly go up sample, and with the 2 times of initial moving phase flushing of column volume chromatography columns, carry out wash-out with initial mobile addition 0.01-0.5NaCl gradient then, collect target peak.Add solid (NH in the ion-exchange target peak ₄) ₂SO ₄To 0.6mol/L, last Phenyl-Sepharose FF post with 20mM pH8.0Tris-hydrochloride buffer gradient elution, is collected target peak, adds solid (NH ₄) ₂SO ₄To 2.0mol/L, 4 ℃ of preservations are spent the night, and 10,000rpm, the 10min centrifugal collecting precipitation, with the dissolving of 10mM pH7.0 phosphate buffered saline buffer, last through same phosphate buffered saline buffer equilibrated Superdex75 post, wash-out is collected target peak.

Claims (5)

1. the production method of a neurotrophic factor, it is characterized in that passing through computer aided design (CAD), gene to Human Ciliary Neurotrophic Factor is modified transformation, obtain to transform the cDNA of back sudden change Ciliary Neurotrophic Factor Gene, this cDNA of synthetic, construction recombination plasmid, transformed into escherichia coli is expressed through the automatic fermenter high-density; Carry out renaturation in the cross-flow ultrafiltration mode; With ion exchange chromatography, hydrophobic chromatography and sieve chromatography sequential combination purifying, produce and obtain highly purified ciliary neurotrophic factor.

2. the production method of a kind of neurotrophic factor according to claim 1, it is characterized in that said Ciliary Neurotrophic Factor Gene modifies that to transform be that primary structure to its coding changes: the 17th halfcystine becomes L-Ala, the 63rd L-glutamic acid becomes arginine, and removes terminal 15 amino acid.

3. the production method of a kind of neurotrophic factor according to claim 2 is characterized in that it is to have selected for use the transformation of intestinal bacteria preference codon to modify that said primary structure to its coding has carried out changing, and makes it obtain high expression level.

4. the production method of a kind of neurotrophic factor according to claim 1 is characterized in that the ultra-filtration membrane material of said cross-flow ultrafiltration adopts low protein adsorbents such as acetyl cellulose, cellulose nitrate ester, inclined to one side tetrafluoroethylene or polysulfones; Molecular weight cut-off is between 1000-10000.

5. the production method of a kind of neurotrophic factor according to claim 1, it is characterized in that said ion exchange chromatography, hydrophobic chromatography and sieve chromatography intermediate ion exchange filler is that DEAE, Q anion exchange groups, dewatering filling are the medium of butyl-, phenyl-or octyl base, molecular sieve filling is the Sephacryl S200 or the Superdex75 of low protein adsorption.