CN106084030B - A method of improving interleukins IL-34 thermal stability - Google Patents

Abstract

The present invention relates to a kind of methods for improving interleukins IL-34 thermal stability, belong to field of biotechnology.The method is by the way that the codon agc for encoding No. 98 serines in source of people IL-34cDNA, rite-directed mutagenesis is the codon tgt of encoding aminothiopropionic acid；Transferring plasmid containing S98C IL-34 mutant code sequence and BacVector-3000 baculovirus DNA cotransfection Sf9 insect cell are prepared to the baculoviral of expression S98C IL-34；Reuse baculoviral transduction 293 cell of HEK expression S98C IL-34.Compared with native IL-34, thermal stability gets a promotion the S98C IL-34 that the method obtains.By M-NFS-60 cell proliferation experiment and CSF-1R phosphorylation assays, it was demonstrated that S98C IL-34 activity and native IL-34 do not have marked difference.

Description

A method of improving interleukins IL-34 thermal stability

Technical field

The present invention relates to a kind of sides of raising interleukins IL-34 (interleukin-34, IL-34) thermal stability Method belongs to biotechnology neck specifically, being related to a kind of method for promoting IL-34 thermal stability by Amino Acid-Induced Site-Directed Mutation Domain.

Background technique

Interleukin 34 (interleukin-34, IL-34) is a newly discovered cell factor (Haishan Lin et al.Science 2008).It is colony-stimulating factor -1 receptor (colony-stimulating factor Lreceptor, CSF-1R) a new ligand, it can specific recognition and discrete actuation CSF-1R.IL-34 plays bioactivity Form be dimeric forms, the molecular weight 50KD of biological dimer, the molecular weight of each monomer is 25KD, the amino acid of monomer Sequence are as follows:

IL-34 is since being identified as the new ligand of CSF-1R, its function and structure is by in-depth study. Although IL-34 has the effect of activation CSF-1R similar with CSF-1, but activation effect is stronger, half-life period shorter (Eda H et al.Cytokine.2010).IL-34 can be secreted in many tissues, but is leading (Clavel G et with the secretion of spleen al.Joint Bone Spine.2013).IL-34 can promote the survival of monocyte, be proliferated and be divided into macrophage. IL-34 can also promote osteoclast to generate.Some researches show that the horizontal phases of IL-34 in patient with rheumatoid arthritis joint fluid There are promotion, while the serious journey of expression and inflammation of the IL-34 in synovial membrane and joint fluid than general arthritic Degree is positively correlated (Baud ' huin M et al.J Pathol 2010；Hwang S-J et al.ArthritisRes Ther 2012)。

Although IL-34 has the ability of activation CSF-1R similar with CSF-1, research points out that their function is not heavy Folded, there are space and time difference (Wei, S.et al.J.Leukoc.Biol.2010) for their expression way.Tetsuya Mizuno reports that IL-34 is capable of repair ability (T of the enhancing mouse microglia for neurotoxicity of selectivity Mizuno.et al.Am J Pathol.2011).There is research to pass through IL-34 simultaneously^LacZ/LacZMouse model, discovery IL-34 tool Organized limited (Wang, Y.et al.Nat Immunol.2012).The research finds IL-34 mainly by skin and maincenter mind Through system secretion, in IL-34 missing, the Langerhans cell development of epithelium is affected and mouse is made contact hypersensitivity occur Shape, and the microglia cell for lacking IL-34 can quantitatively be affected, and weaken microglia for central nervous system The protective capability of system.This research shows IL-34 has patent medicine potentiality in terms of neuroprotection and repair.

Significant influence that IL-34 shows in neuron reparation so that IL-34 be provided with patent medicine possibility (Wang, Y.et al.Nat Immunol.2012).For protein medicaments, can shelf-life and half-life period in vivo patent medicine Essential condition, these conditions all have relationship with the thermal stability of albumen.If a new cell factor will become marketed drug, It has to be promoted the stability of albumen, but there is no the method for promoting IL-34 thermal stability since self-discovery.

Summary of the invention

In view of the defects existing in the prior art, the purpose of the present invention is to provide a kind of sides for improving IL-34 thermal stability Method, the method improve the thermal stability of IL-34 by carrying out Amino Acid-Induced Site-Directed Mutation to IL-34, and improving, IL-34 heat is steady While qualitative, the bioactivity of IL-34 is maintained, allows IL-34 that there are bigger patent medicine potentiality.

The purpose of the present invention is what is be achieved through the following technical solutions.

A method of IL-34 thermal stability being improved, the method, will by carrying out Amino Acid-Induced Site-Directed Mutation to IL-34 No. 98 mutant serines of IL-34 improve the thermal stability of IL-34 at cysteine, hereinafter, are referred to S98C IL-34 No. 98 serine residues are mutated into the IL-34 of cysteine, and the amino acid sequence of the S98C IL-34 monomer is as follows:

Specific step is as follows for the method:

The password of No. 98 serines will be expressed in the IL-34 cDNA of the expression protein of GeneBank ID:NM_152456 Sub- agc uses site-directed point mutation for the codon tgt of expression cysteine；By after mutation S98C IL-34 cDNA with BacVector-3000 baculovirus DNA cotransfection Sf9 insect cell expresses the baculoviral of S98C IL-34 to produce；Make again S98C IL-34 is expressed with 293 cell of baculovirus infection HEK.

Beneficial effect

1. the present invention provides a kind of method for improving IL-34 thermal stability, the method is based on existing IL-34 structure (PDB ID:4DKC) analysis, discovery at protein core No. 98 serine residues and No. 168 cysteine residues apart from close, The distance of the two C α isIn order to promote the thermal stability of IL-34, No. 98 serine residues of IL-34 are mutated into half Guang Propylhomoserin, hereafter refers to unmutated IL-34 with native IL-34, and S98C IL-34 refers to No. 98 serine residues and is mutated into half The IL-34 of cystine；

2. the present invention provides a kind of method for improving IL-34 thermal stability, the method passes through albumin crystal technology knot Brilliant S98C IL-34 albumen simultaneously parses its structure, confirms that S98C IL-34 is mutated successfully from crystal structure level, and confirm 98 Number the disulfide bond that native IL-34 does not have is formd between cysteine and No. 168 cysteines；

3. the present invention provides it is a kind of improve IL-34 thermal stability method, the method obtain S98C IL-34 with Native IL-34 is compared, T_m(melting temperature) improves 7.6 DEG C, illustrates S98C IL-34 compared to native IL-34 thermal stability is improved.Simultaneously by the phosphorylation assays of the proliferation experiment of M-NFS-60 cell and CSF-1R, card The biological activity and native IL-34 of bright S98C IL-34 does not have marked difference.

Detailed description of the invention

Fig. 1 is that schematic diagram is arranged in PCR program.

Fig. 2 is the SDS-PAGE qualification figure of Native IL-34 and S98C IL-34.

Fig. 3 is that Native IL-34 and S98C IL-34 scheme through molecular sieve system purifying.

Fig. 4 is obtained S98C IL-34 crystal after condition optimizing.

Fig. 5 is S98C IL-34 crystal structure (left side) and native IL-34 crystal structure figure (right side).

Fig. 6 is the Fo-Fc differential electronic density of Cys98-Cys168 disulfide bond in the monomer of S98C IL-34 crystal structure Figure is (left: A chain；It is right: B chain；Fo-Fc level=3.0).

Fig. 7 is the synchrotron radiation CD map of native IL-34 at different temperatures.

Fig. 8 is the synchrotron radiation CD map of S98C IL-34 at different temperatures.

Fig. 9 is the two condition inversion equilibrium fitted figure of Native IL-34 and S98C IL-34.

Figure 10 is the concentration dependency curves figure that S98C IL-34 and native IL-34 stimulate M-NFS-60 cell Proliferation.

Figure 11 is that S98C IL-34 and native IL-34 activation generate the CSF-1R (pCSF-1R) of phosphorylation and intracellular The Western blot histogram of overall CSF-1R (Total CSF-1R).

Figure 12 is that S98C IL-34 and native IL-34 activation generate the CSF-1R (pCSF-1R) of phosphorylation and intracellular The gray level ratio histogram of the Western blot band of overall CSF-1R (Total CSF-1R).

The amino acid alignment figure that Figure 13 is the IL-34 of different genera (refers to Uniprot database, with individual character matrix Show amino acid).

Specific embodiment

Embodiment 1

1. the building of expression plasmid

Material:

The cDNA of IL-34 is purchased from Origene company.Used cloning vector is BacMam carrier.

Experiment strain Escherichia coli used is DH5 α bacterial strain, is purchased from Ding Guo biotech firm.

PCR primer is won Radix Polygalae company by Beijing three and is synthesized.

PCR related reagent: Vent archaeal dna polymerase (NEB), dNTP mixture (NEB), MgSO4 (NEB), DMSO (NEB), 10 × PCR buffer (NEB).

DNA clone related reagent: agarose (invitrogen), restriction enzyme (NEB), T4 DNA ligase (NEB), gel reclaims kit (Promega), the small extraction reagent kit of plasmid (Promega), nucleic acid dye GelRed (Biotium)。

Bacteria Culture reagent: yeast extract (OXOID), peptone (OXOID), ampicillin (Amresco), LB- Agar(Sigma-Aldrich)。

Experiment is prepared with ultrapure water by Milli-Q pure water system.

Method and result:

(1) the amplification gene segment from cDNA

This step mainly uses polymerase chain reaction (PCR) to expand and rite-directed mutagenesis IL-34cDNA, to generate The gene of native IL-34 and S98C IL-34.The reaction system total volume of PCR experiment is 50 μ L.System component such as table 1, Such as Fig. 1 shows PCR primer is shown in Table 2 to the setting of PCR program.

1 PCR reaction system component of table

2 PCR primer of table

(2) the recycling purification of PCR product

After PCR experiment, 5 μ l reaction products is taken to carry out agarose gel electrophoresis, to determine whether PCR reaction successfully expands Increase purpose band.The setting parameter of agarose gel electrophoresis is voltage 100V, electrophoresis time 30min.

After agarose gel electrophoresis, the purpose base in product is extracted using the PCR product QIAquick Gel Extraction Kit of Promega company Cause.Extraction step is carried out according to kit specification.

(3) the double limitation digestions and connection of target gene

Use the product and vector plasmid BacMam of restricted shearing enzymatic treatment after purification.Enzyme when according to design of primers Enzyme site selects corresponding restricted shearing enzyme, and restricted shearing enzyme used in experiment includes BamHI, NotI, XhoI etc., reaction System such as table 3.

3 pairs of limitation ligases of table test component

Endonuclease reaction system is placed in 37 DEG C of water-bath 3h.Then the good purpose of digestion is obtained using agarose gel electrophoresis to produce Object band and vector plasmid band.The target gene and load in gel are extracted using the DNA QIAquick Gel Extraction Kit of Promega company Constitution grain.Extraction step is carried out according to kit specification.

After double restricted digestion processing, target gene is connect with vector plasmid, reaction system is shown in Table 4.Reaction is in room temperature Lower carry out 60min.

The reactive component that 4 target gene of table is connect with carrier

(4) conversion of connection product

Connection product is added in -80 DEG C of refrigerators after taking out and the 100 μ l DH5 α competent cells just melted are added In, 30min is stood on ice.Then the heat shock 90s in 42 DEG C of water-baths, is transferred quickly to place 2min or so on ice.Ultra-clean 800 μ l SOC culture mediums are added in platform, are put into 37 DEG C, recovery 40min in 5rpm shaking table.Finally by bacterium solution be uniformly coated on added with Resistance screening is carried out on the LB plate of ampicillin, 14~16h is placed in 37 DEG C of incubators.

(5) screening of positive colony

Picking single colonie is inoculated in the 3ml LB culture medium added with ampicillin, is put into 37 DEG C, 180rpm shaking table 12~14h of middle culture.Plasmid is extracted using the small extraction reagent kit of Promega plasmid, and carries out digestion identification, choosing qualification result is Positive clone carries out gene sequencing, and examining order sequencing company is completed.The correct plasmid of sequencing result is stored in -20 DEG C of ice It is spare in case.

2. the expression of recombinant protein

Material:

The cell line that virus amplification is used in the present embodiment is sf9 insect cell, and the cell line for expressing albumen is HEK293 cell.

Eukaryotic cell lines of the table 5 for virus preparation and protein expression

Insect cell medium includes: Grace, SF-II 900 (Gibco)

Mammalian cell culture: DMEM (Gibco), fetal calf serum (Gibco), mycillin (Gibco)

Transfection reagent: Cellfectin (Invitrogen)

Method:

(1) insect cell preparation and reorganization baculoviral is utilized

1. the sf9 cell of logarithmic growth phase, is dyed using trypan blue staining.When survival rate 98% (i.e. 98% with On cell not by Trypan Blue) when, take sf9 cell to be added in 6 orifice plates, allow total number of cells about 1.0 × 10 in every hole⁷ A, volume is about 2ml.6 orifice plates are put into 27 DEG C of incubators and stand adherent 1h.

2. the 6 μ l of plasmid for taking sequencing positive is mixed with 2 μ l BacVector-3000 baculovirus DNAs, it is incubated for 5min.It takes 10 μ l Cellfectin transfection reagents are uniformly mixed with 100 μ l SF900 Insect culture medium without double antibody.By above two mixed liquor It is uniformly mixed, is incubated for 30min.

3. after sf9 cell is adherent, discarding former culture medium, sf900 culture medium without double antibody is changed into, and be added mixed in step 2 Close liquid.It is put into stationary culture 6h in 27 DEG C of incubators.

4. after 6h, the culture medium without double antibody in six orifice plates to be substituted for dual anti-sf900 culture medium.Continue to train at 27 DEG C Support stationary culture in case.

5. after 7d, harvesting the supernatant in every hole, the P1 virus of plasmid is as corresponded to.P1 virus titer is low, is mainly used for expanding Increase P2 virus or be stored in -80 DEG C of refrigerators and saves.

6. P1 virus to be infected to the sf9 cell of logarithmic growth phase by 1: 1000 volume ratio.Allow cell in 27 DEG C of incubators In continue shaking culture.

7. infecting about 4d or so, when observing cell growth retardation, cell becomes larger, and about half cell disruption Afterwards, the supernatant of cell is harvested to get P2 virus, is stored in 4 DEG C in case using.

8. if it is desired, it is viral to prepare large batch of P3 according to step 6,7 to can use P2 virus.

(2) mammalian cell expression albumen is utilized

This step will utilize human embryonic kidney cell, i.e. HEK293 cell expresses albumen.

1. being about 3.0 × 10 by HEK293 cell culture to density⁶A/ml, and ensure that cell is deposited with trypan blue staining Motility rate is 90% or more.

2. after 72h, centrifugation discards cell by the suitable addition HEK293 cell of P2 or P3 virus, supernatant culture is collected Base.HBS buffer is concentrated and is continuously added to culture medium using cross-flow ultrafiltration system, make the environment of destination protein from DMEM culture medium exchanges to HBS buffer.

3. the destination protein solution after exchange concentration is used supercentrifuge, 13000g is centrifuged 20min, molten to remove Cell fragment in liquid.

(3) destination protein is extracted using affinity chromatography

6 histidine tags (- HHHHHH) that this step is added using C-terminal when design albumen, using Ni-NTA agar Sugared gel particle is stationary phase, carries out elution purification to destination protein.

1. 4 DEG C are incubated for 1 in the protein solution agarose gel particle of appropriate coupling Ni-NTA being added to after high speed centrifugation ~2h.

2. clean Ni-NTA agarose gel particle with the cleaning buffer solution containing 20mM imidazoles, with wash away not with Ni-NTA The non-destination protein of complexing.

3. the elution buffer containing 40mM and 300mM imidazoles is used to wash Ni-NTA agarose gel particle It is de-, eluent is collected, 4 DEG C of refrigerators is placed in and saves.

(4) SDS-PAGE testing goal albumen is utilized

This step is different using the distance that the albumen of different molecular weight advances in SDS-PAGE protein electrophoresis, to identify Whether the albumen of elution is purpose albumen.

1. taking 5 μ l that 5 × sample-loading buffer is added through the protein sample of affinitive layer purification, mix, 95 DEG C of heating 5min, egg It is cooling in room temperature after white abundant denaturation.

2. quantitatively drawing the protein sample being denaturalized through SDS with micro sample adding appliance, it is added in the well of running gel.

3. voltage 140V, electrophoresis time about 90min is arranged, when electrophoresis to bromophenol blue indicator reaches gel lower edge, stop Electrophoresis.

4. carefully removing gel, it is put into the vessel for filling coomassie brilliant blue R_250 dyeing liquor, covers ware lid, in shaking table On in room temperature stained over night.

5. gel is moved into the vessel for filling destainer, decoloration is vibrated on horizontal oscillations shaking table at room temperature, until Gel clean background is transparent, until protein band colour developing is good.

6. identifying whether gained albumen is purpose albumen according to the instruction of albumen Marker.

As a result:

Native IL-34 and S98C IL-34 are expressed successfully, and monomer molecule amount is 25KD, meet expection, SDS-PAGE knot Fruit is referring to fig. 2.

3. protein purification is identified

Material:

0.22 μm of filter membrane (PALL)

Ultra-filtration centrifuge tube (molecular cut off 10000) (Merk)

GE AKTA FPLC protein purification system

GE superdex-200 analysis sieve gel chromatography column

Method:

(1) after the identification of SDS-PAGE, identical albumen is merged.The ultrafiltration for the use of molecular cut off being 10000 Destination protein is suitably concentrated centrifuge tube, to reduce the loading number of molecular sieve system.

(2) protein sample being concentrated is centrifuged with high speed freezing centrifuge, parameter of noncentricity is 4 DEG C, 13000rpm, centrifugation Time is 10min.

(3) prepare the mobile phase of sieve purification system.It is all for the ultrapure water of sieve purification system, buffer, And then storing liquid (20% ethyl alcohol), the membrane filtration for being 0.22 μm with aperture are evacuated 10min with vacuum pump, to drain liquid Intracorporal gas.

(4) after GE superdex-200 being analyzed the upper purification system of sieve column connection, setting system flow rate is 0.5ml/min, Upper pressure limit is 1.5MPa, using one column volume of ultrapure water, reuses HBS elution buffer and balances a column volume.

(5) before protein sample loading, first use HBS buffer solution for cleaning loading ring, after loading, system is allowed to start to elute, if It sets and connects that sample volume is 1ml, elution volume is 1.2 column volumes.

(6) refined solution being collected into is identified whether albumen purifies success by SDS-PAGE experiment is carried out.

As a result:

Native IL-34 and mutant S98C IL-34 can be purified by sieve purification system, and eluting peak concentrates on 15~17ml, referring to Fig. 3.

4.S98C IL-34 albumin crystal structure determination

Material:

Crystallization condition screening reagent box: including this five quotient of Hampton, Wizard, Proplex, Memstart, Memsys With screening reagent box.Each kit includes 96 kinds of screening conditions.

For configuring the classes of agents of crystallization condition optimization:

NaCl (Sigma-Aldrich), Na-Citrate (Sigma-Aldrich), LiSO₄(aladdin), Na₂HPO₄ (Chinese medicines group), KH₂PO₄(Chinese medicines group), PEG series (Sigma-Aldrich), CHES (Sigma-Aldrich), Tris (Genview), (NH₄)₂SO₄(Alfa Aesar), NaAc (Amresco), MgCl₂(Sigma-Aldrich), CaCl₂(traditional Chinese medicines collection Group).

Method and result:

Using crystallization condition screening reagent box, the crystallization condition of S98C IL-34 is screened at 20 DEG C with sessile drop method.To S98C After IL-34 crystallization, optimized according to the crystallization condition of screening, the crystallization condition of optimization such as table 6, the protein of S98C IL-34 Crystal such as Fig. 4.

The optimal conditions of 6 6017 crystal of table

The collection work of crystal diffraction data is completed in Beijing Institute of High-energy Physics Technology Synchrotron Radiation (BSRF).With HKL2000 software handles diffraction data, parses crystal structure with CCP4 software.Obtain the crystal structure of S98C IL-34 with The structure of native IL-34 does not have notable difference (Fig. 5).Fo-Fc differential electronic density map shows No. 98 of S98C IL-34 Residue has been mutated into cysteine, and forms disulfide bond (Fig. 6) with No. 168 cysteine residues.

5. synchrotron radiation light source circular dichroism detector (CD) measures thermal stability

Instrument and material:

(1) instrument

Instrument used in synchrotron radiation circular dichroism spectra is located at 4B8 vacuum ultraviolet light beam line (the BSRF 4B8 VUV of BSRF Beamline), the vacuum ultraviolet and ultraviolet light, 4~90 DEG C of temperature range which can provide 125~360nm are for justifying two Chromatography test.

The quartz sample pool that experiment centre provides includes different light path: 0.01mm, 0.1mm, 0.2mm, 1mm.Wherein light path The sample cell of 1mm, volume are 350 μ L.The sample cell of light path 0.1mm, volume are about several tens of microliters.In experiment, what we used It is the sample cell that light path is 0.1mm.

The control acquisition software of experiment centre circular dichroism spectra instrument is BSRF 4B8 VUV Station.Pass through the control of software Interface, can directly select circular dichroism spectra scanning starting and terminate wavelength, the interval of scanning wavelength, the number scanned every time with And constant temperature time etc., therefore parameter can be set by control software and carry out automatically continuous Caloric test.

(2) laboratory sample

Experiment is native IL-34 and S98C the IL-34 recombinant protein of expression.

NaCl original in PBS is replaced with NaF when preparation of samples, to reduce Cl^-Interference for circular dichroism spectra.

Method:

(1) sample is filled

Sample cell light path used in experiment is 0.1mm.

Sample cell is formed by stacking by two panels circular quartz glass.For two pieces of quartz glass when covering conjunction, the region of label is stringent It fits together.It is finished in dress sample, after two panels sample cell glass is by demarcating position folded and strictly confirming no bubble, by sample Pond is packed into specimen holder.

When sample cell is packed into specimen holder, the outside groove of the congruent points counter sample frame of sample cell.It then will be assembled Specimen holder is put into sample room, covers the insulation cover of sample room later, and closes the lid of sample room.

(2) spectral measurement

The concentration that solution component in experiment is native IL-34 and S98C IL-34 is respectively 80 μM, denaturant hydrochloric acid The concentration of guanidine is 1M, and solution buffer system is PBS, and the NaCl in PBS is replaced by NaF.

During alternating temperature, 10 temperature spots are set, are respectively as follows: 25 DEG C, 35 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, reach the minimum point progress one-shot measurement that maximum temperature point carries 25 DEG C of return.When each temperature spot constant temperature Between be 5 minutes, allow albumen sufficiently to heat.The wave-length coverage measured every time be 200nm~260nm, sweep spacing 1nm, each Temperature spot measures three times.

As a result:

All readings measured three times of the same temperature of synchrotron radiation circular dichroism spectra instrument record, therefore at the same temperature Three groups of experimental datas are had, the initial data image tested is shown in Fig. 7 and Fig. 8.

Choose the calculated unfolding ingredient F of ellipticity of the 222nm in initial data_UFor dependent variable (equation A), temperature For independent variable, two condition inversion equilibrium formula (Jackson.et al.Biochemistry.1991 is used；Nicholson.et al.Biochemistry.1996；Equation B) carry out the T of fitting n ative IL-34 and S98C IL-34_m。

Wherein, [θ]_NAlbumen is in the CD value under 100% folded state when for initial temperature, and [θ] is under different temperatures CD value, [θ]_DFor albumen 100% denaturation unfolding CD value.

Wherein, R is ideal gas constant, and R=1.987cal/ (Kmol), T are temperature locating for protein, T_mFor egg White matter melting temperature during thermal denaturation, Δ H_mT is in for temperature_mWhen protein enthalpy change.

The solving result of the two condition Equation of 7 native IL-34 and S98C IL-34 of table

Fitting result such as table 7, matched curve is referring to Fig. 9.

According to the Fitting Calculation as a result, the T of native IL-34_mIt is 50.9 DEG C, the T of S98C IL-34_mIt is 58.5 DEG C, S98C The T of IL-34_m7.6 DEG C are improved compared to native IL-34, illustrates the thermal stability of the mutant S98C IL-34 of the present embodiment Better than native IL-34.

6. cell proliferation experiment

Material:

M-NFS-60 cell

Divine Land is stuck up purchased from Gibco and justice for M-NFS-60 cell recovery and the rhCSF-1 of passage.The culture used Based formulas are as follows:

1640 culture medium of PRMI (Gibco)

10% fetal calf serum (Gibco)

100U/ml penicillin&streptomycin(Gibco)

60ng/ml rhCSF-1

Method:

(1) it is cultivated with the culture M-NFS-60 cell that suspends of the culture medium containing rhCSF-1 to logarithmic phase, then in no rhCSF-1 It is 24 hours hungry in base；

(2) by cell according to every hole 0.1 × 10⁶It is a to be added to 96 holes entirely in opaque blank；Cell is divided into four groups, only adds PBS is negative control group, and rhCSF-1 is positive controls, and experimental group is native IL-34, two groups of S98C IL-34.It is added The concentration gradient of albumen are as follows:

10^-3, 10^-2, 10^-1, 10⁰, 10¹, 10², 10³, 10⁴, 10⁵(ng/ml)

(3) cell is put into 37 DEG C of carbon dioxide incubators suspension cultures；Using at CellTiter-Glo kit after 3d Cell is managed, and uses Flexstation fluorescence reader measurement relative fluorescence readings (relative light units, RLU).

The RLU of cell sample is respectively to the dose-dependence curve of rhCSF-1, native IL-34, S98C IL-34 As shown in Figure 10；Extrapolate the EC that three promotees cell Proliferation₅₀Value is as shown in table 8.

Table 8

Wherein, the EC of native IL-34, S98C IL-34₅₀Concentration is respectively 1066ng/ml and 973ng/ml explanation The ability that S98C IL-34 and native IL-34 promote M-NFS-60 cell Proliferation is close.

The comparison of 7.S98C IL-34 and native IL-34 activation CSF-1R ability

Material:

The host cell that transfection expression overall length CSF-1R is used in this step is HEK293 cell.

Primary antibody is rabbit source CSF-1R antibody (anti-CSF-1R), rabbit source phosphorylation CSF-1R antibody (anti- PhosphoCSF-1R-Tyr723), it is purchased from CST.

Secondary antibody is goat anti-rabbit IgG antibody (the green skies).

Cell pyrolysis liquid (the green skies)

Method:

(1) the HEK293 cell of normal growth in 37 DEG C of carbon dioxide incubators is taken, equivalent is divided into six orifice plates, and every hole is thin Born of the same parents' number is 1.0 × 10⁶.Equivalent and suitable CSF-1R baculoviral are added in every hole.

(2) every hole cell is washed twice of cell using serum-free DMEM after 72h, and cell is placed in famine in serum-free DMEM Starve 16h.

(3) cell is divided into three groups: experimental group is native IL-34 and S98C IL-34, and albumen is added to its end in every hole Concentration is 5 μ g/ml；PBS is only added in negative control group cell.

(4) cell is centrifuged after 5min, and is washed three times with 4 DEG C of PBS, then crack 30min on ice in lysate.

(5) take cell pyrolysis liquid in quantitative on Nanodrop protein quantification instrument, it is ensured that the protein content in each loading hole is identical, Then SDS-PAGE electrophoresis is carried out.

(6) after SDS-PAGE, Western blot processing is carried out to running gel.Wherein primary antibody (anti-CSF-1R and Anti-phosphoCSF-1R-Tyr723) dilution is 1: 1000, is incubated for 2h；Secondary antibody dilution is 1: 2000, and incubation time is 50min.Then it is exposed processing, collects image result.

As a result:

Figure 11 and Figure 12 shows the energy of S98C IL-34 and native IL-34 mutant activation CSF-1R autophosphorylation Power does not have notable difference, to illustrate that S98C IL-34 and native IL-34 has similar biological activity.

The conservative Analysis of 8.S98C IL-34 intramolecule disulfide bond Cys98-C168

As shown in the gray area of Figure 13, the Ser98 and Cys168 of source of people IL-34 (Uniprot ID:Q6ZMJ4) is at it IL-34 (mouse Q8R1R4, ox A6QL48, rat Q4KM46, zebra fish L8AZT5, the chimpanzee H2QBG6, cat of his species M3XD55, giant panda G1L622) in absolute conservation.It is same with 98 and No. 168 residues of source of people IL-34 in other species IL-34 Disulfide bond is introduced on the position in source, to improve the way of its thermal stability, is considered as and introduces two in S98C IL-34 intramolecule The principle that sulfide linkage Cys98-C168 improves thermal stability is identical, belongs to the scope of protection of the present invention.

Although combining attached drawing describes embodiments of the present invention, it will be apparent to those skilled in the art that not Under the premise of being detached from the principle of the invention, several improvement can also be made, these also should be regarded as belonging to the scope of protection of the present invention.

Claims (2)

1. a kind of method for improving interleukins IL-34 thermal stability, it is characterised in that: the method passes through to source of people IL- 34 carry out Amino Acid-Induced Site-Directed Mutation, by No. 98 mutant serines of source of people IL-34 at cysteine, i.e. S98C IL-34, S98C The amino acid sequence of IL-34 is as follows:

2. a kind of method for improving interleukins IL-34 thermal stability, it is characterised in that: the method passes through baculoviral- Mammalian cell expression system prepares IL-34 mutant, in the method, by the expression egg of GenBank ID:NM_152456 The codon agc that No. 98 serines are encoded in the IL-34 cDNA of white matter, uses site-directed point mutation for encoding aminothiopropionic acid Codon tgt；By the S98C IL-34 cDNA clone after mutation to transfer vector BacMam, the transferring plasmid of acquisition with BacVector-3000 baculovirus DNA cotransfection Sf9 insect cell, to prepare the recombinant baculovirus of S98C IL-34；Again S98C IL-34 is expressed using recombinant baculovirus transduction 293 cell of HEK.