CN101045923A - Process of producing interleukin analog - Google Patents

Abstract

The colibacillus expression and hydrophobic chromatographic purification to produce protein with amino acid sequence similar to that of human interleukin-2 can obtain protein with specific activity after being purified not lower than 1x108 IU/mg.

Description

Produce a kind of method of interleukin analog

Technical field

The present invention relates to a kind of human interleukin-12 analogue and utilize genetic engineering technique to produce the method for this analogue.

Background technology

Interleukin II (also claim the T cell growth factor, this paper is called for short IL2) is a kind of lymphokine that the T cell produces under lectin or isoantigen stimulation, and IL2 can make the T cell keep function constant when external long term growth.IL2 promotes thymocyte mitotic division in addition, makes nude mice spleen cells regenerate the T cell and relies on antigenic antibody (the T cell is replaced the factor), promotes killer cell differentiation and propagation (killing and wounding cofactor).Utilized IL2 to clone killer T cell clone, helper cell clone and natural killer cell clone.Except that direct application and T cell or natural killer cell clone, IL2 can be used for the selective growth of the killer T cell of certain antigen-specific, and for example killer T cell is at a kind of tumour antigen of external identification and destroy tumour cell.The tumour-specific killer T cell that injection utilizes IL2 to stimulate in animal body may suppress and prevent tumor growth.IL2 can also stimulate IFN γ to generate and activate natural killer cell.

Above experimental data shows that IL2 can be used as a kind of anti-tumor agent.IL2 can recover to lack the function of helper cell in the nude mice of thymus function or recover the killer T cell of homogeneous cell induction, so IL2 can be used for the treatment of immunological disease.

Recombinated interleukin-2 is treated diseases such as carcinous pleural effusions and ascites and kidney, melanoma clinically and has been applied, along with going deep into of using, present deficiency gradually: 1) dosage is on the low side, curative effect is not remarkable, the recombinant human interleukin--2 causes clinical effectiveness not obvious because of problems such as its hydrophobicity are difficult to make the high dosage goods; 2) foreign matter content is higher, contain three halfcystines (Cys) in the coded rhIL-2 albumen primary structure of natural hIL-2 gene order, lay respectively at 58,105 and 125, wherein the disulfide linkage of the sulfydryl of the 58th and 105 Cys formation is most important to the rIL-2 biologic activity ¹²⁵The sulfydryl of Cys is unbound state, and is very unstable, can form impurity with 58 or 105 sulfydryl mispairing in some cases, and IL-2 is active to be reduced thereby make, and influences the medication effect.

125 in the IL2 sequence are carried out the amino acid transformation, for example original C ys are replaced with L-Ala or Serine, improved amino acid not can with the 58th or 105 Cys mispairing, reduce foreign matter content greatly, this series products to be used clinically.In addition, the eighties consonance fermentation in last century Co., Ltd. discloses the biological activity that can improve IL2 in proteic the 1st to the 5th amino acids disappearance of IL2.Therefore a kind of N end disappearance and have higher biological activity and lower foreign matter content at the interleukin II analogue of the 125th sudden change.

The method for preparing interleukin II by bacterial expression is known.Ordinary method is expressed the back with the form of inclusion body or directly be present in the tenuigenin based on protein in intestinal bacteria (E.Coli).For the interleukin II that exists with the inclusion body form, owing to can not correctly folding, protein molecular do not have biological activity, must in cell, isolate inclusion body, adopt high density denaturing agent dissolving inclusion body, remove the concentration of denaturing agent or reduction denaturing agent then, make inclusion body protein be able to renaturation, carry out step purifying such as ion exchange chromatography, affinity chromatography, RPHPLC (reversed-phase high-performance liquid chromatography), molecular exclusion chromatography again and could obtain can be used for clinical goods.Wherein the renaturation of inclusion body protein and purifying are the cores in the whole process.

Ubiquitous problem is in the recombinant protein production at present: (1) annealing efficiency is low.Traditional refolding method comprises dilution method and dialysis method.The dilution refolding method is to tens times in sample, even the dilution of hundreds of times can make the volume of sample sharply increase, and brings very big difficulty for follow-up separation and purification, and needs bigger renaturation container in the renaturation process.Dialysis method is consuming time longer, and will repeatedly change dialysis solution.The common drawback of these two kinds of methods is that protein can take place to assemble and a large amount of precipitations of generation in renaturation process, annealing efficiency is low, usually the activity of proteins rate of recovery has only 5～20%, and contains a large amount of foreign proteins in the protein soln after the renaturation, need carry out further separation and purification.(2) production cost height, facility investment is big.Because renaturation and separation and purification are carried out separately respectively, and purification procedures is many, each step all needs supporting with it equipment, causes facility investment big, the production cost height.Along with production-scale increase, this drawback can be more and more serious.

The early 1990s in last century, China at first has report efficient hydrophobic interaction chromatograph (HPHIC) to be used for the renaturation of metaprotein, well solve the problems referred to above, now successfully be used for the purifying process of recombinant proteins such as recombinant human interferon gamma, recombinant methionyl human G-CSF, reorganization bovine prion protein.After this, exclusion chromatography, ion exchange chromatography and affinity chromatography also are useful in proteinic renaturation and the while purifying.But the using hydrophobic chromatography is not appeared in the newspapers as yet to the method that the interleukin II analogue carries out renaturation and purifying simultaneously.Hydrophobic chromatography use a protein surface part have hydrophobicity, under high salt concn with gel skeleton on crosslinked butyl, phenyl bonded principle.

Compare with traditional dilution method and dialysis method, the advantage of carrying out interleukin II analogue renaturation with hydrophobic chromatography is: 1. can remove denaturing agent very soon behind sample introduction; 2. owing to the absorption of hydrophobic grouping on the gel skeleton, can reduce even eliminate fully protein aggregate and sedimentary generation in the renaturation process significantly, thereby improve the quality of protein renaturation, significantly reduce aggressiveness and monomer foreign matter content denatured protein; Target protein is separated with foreign protein to reach the purpose of purifying, renaturation and purifying are carried out simultaneously; 4. be convenient to reclaim denaturing agent, to reduce cost for wastewater treatment.In brief, the hydrophobic chromatography renaturation can improve the interleukin II analogue purity, reduce foreign matter content, protein renaturation and purifying are integrated in single stepping finish, shortened operation steps and production time, reduced facility investment, production cost is reduced greatly.

Summary of the invention

The purpose of this invention is to provide a kind of human interleukin-12 analogue and utilize genetic engineering technique to produce the method for this analogue.The present invention further provides the method that a kind of method of using on-column refolding prepares the interleukin II analogue.The present invention further provides a kind of purification ratio activity and be not less than 1 * 10 ⁸The method of the proteic interleukin II analogue of international unit/milligram.

Concrete steps comprise: the 1) gene of composite coding interleukin II analogue nucleotide sequence, 2) fermentation of bacterial cell, 3) extraction and the purifying of inclusion body, 4) hydrophobic chromatography renaturation and purifying inclusion body, 5) ion exchange chromatography, 6) gel exclusion chromatography.

The invention is not restricted to use intestinal bacteria, comprise that also use can express any prokaryotic micro-organisms of the heterologous protein of inclusion body form.

Detailed Description Of The Invention

The gene of express recombinant human interleukin-12 analogue utilizes chemical process synthetic.IL2 analogue gene fragment is connected in the commercialization prokaryotic expression plasmid, and these plasmids include but not limited to pTrc, pBAD, pTac, pET, pT7, pL etc.; Recombinant plasmid is transfected into the host bacterium, and these host bacterium include but not limited to DH5 α, JM109, JM103, HB101 etc.

Step 1: fermentation

The bacterial strain that ferments in the inventive method was preserved to keep its ability to express with lyophilize thing form before using.During use,, begin fermentation after being extended to requirement with being inoculated on the solid medium after the dissolving of suitable buffered soln.

Fermentation condition comprises suitable medium, temperature, pH value, dissolved oxygen etc.Fermention medium is the commercialization and the known substratum that can effectively use.The mixed culture medium that preferably contains peptone, yeast extract, glycerine, phosphate-buffered salt etc.Add one or more microbiotic in the substratum.The penbritin of 50-200 μ g/ml preferably.Leavening temperature is 30-42 a ℃ of suitable prokaryotic micro-organisms growth.Among the present invention preferably 37 ℃.PH should remain on neutrality (6.4-7.4) between yeast phase.Tank pressure is a normal pressure in the fermenting process, and dissolved oxygen remains on 20-60%, preferably 50%.Owing under agitation ferment, preferably use defoamer.

In the fermenting process, along with microorganism growth, the optical density(OD) of culture increases.Can monitor fermentation progress degree according to optical density(OD).Usually selecting the wavelength of measuring light density is 600nm.In the method for the present invention, microorganism culturing is begun abduction delivering during to logarithmic phase.Usually microorganism this period 600nm optical density(OD) can reach more than 1, among the present invention preferably optical density(OD) reach 15 and begin to induce when above.According to selected plasmid, the inductive method comprises and adds inductor or change culture temperature.Induction time 2-10 hour, preferably add inductor sec.-propyl-β-sulfo-galactopyranoside (IPTG) among the present invention and induced 3-6 hour.

After inducing end, centrifugal culture is got cell and is carried out the following step.

Step 2: extract and the purifying inclusion body

The interleukin II analogue of expressing is present in the tenuigenin with the form of undissolved inclusion body.Therefore, the invention provides lysis, inclusion body recovery and washing.

Washed cell also is suspended in the suitable damping fluid, preferably contains the neutrality or weakly alkaline (pH6.5-9.0) damping fluid of 0.001mol/L edta and its sodium salt (EDTA) and dithiothreitol (DTT) (DTT).With freeze/melt, French press, supersound process or other similar known technology lysing cell.Add above-mentioned damping fluid in the cell suspending liquid after cracking.Low temperature (2-10 ℃) (10000-15000rpm) centrifugal 10-40 minute at a high speed discards supernatant liquor, and precipitation suspends with above-mentioned damping fluid.Repeated centrifugation and suspension step.The inclusion body that obtains dissolves with the denaturing agent that contains DTT.The 8mol/L urea (pH6.5-9.0) that preferably contains 0.001mol/L DTT among the present invention.

Step 3: the renaturation of inclusion body and purifying

The dissolved inclusion body hydrophobic chromatography post of packing into.Can use any business-like hydrophobic chromatoghaphy medium, as long as the feature class of its capacity, filler and flow velocity is similar to Phenyl Sepharose6 Fast Flow chromatography column, ButylSepharose4 Fast Flow chromatography column, OctyleSepharoe4 Fast Flow chromatography column, Macro-PrepMethyl HIC Support chromatography column, Macro-Prep t-Butyl HIC Support chromatography column etc.The present invention is Butyl Sepharose4 Fast Flow chromatography column preferably.Application contains denaturing agent washing and this chromatographic column of balance of DTT, and renaturation is to realize by the concentration that reduces denaturing agent and in the presence of the oxygenant of proper concn.Among the present invention preferably the linear gradient with 5-20 times of column volume carry out the transition to 2mol/L urea-0.005mol/L Triptide by 8mol/L urea-0.001mol/L DTT, use 0.1mol/L PBS-2mol/L urea (pH7.5) eluted protein at last.Automatically monitor the wash-out of each material by the absorbancy of measuring 280nm.

Step 4: ion exchange chromatography

The component from preceding step that the interleukin II analogue of activity form is rich in collection is packed on the ion exchange column.Can use any commercialization ion-exchange chromatography media, as long as the feature of its capacity, filler and flow velocity is compatible with the method for the invention condition.Preferably SP-Sepharose or CM-Sepharose among the present invention.Use the inorganic salt isocratic elution and can remove partial impurities and target protein is concentrated.The present invention preferably uses 0.1-0.3mol/LNaCl wash-out target protein.

Step 5: gel exclusion chromatography

The component from preceding step that the interleukin II analogue of activity form is rich in collection is packed on the gel exclusion chromatography post.Can use any commercialization gel exclusion chromatography medium, as long as the feature of its capacity, filler and flow velocity is compatible with the method for the invention condition.Sephacyl S-200 preferably among the present invention.Using the characteristic of gel exclusion can remove monomer or aggressiveness impurity and remove remaining organic solvent or inorganic salt.The present invention preferably uses 0.01mol/L phosphate buffered saline buffer wash-out target protein.

The interleukin II analogue e. coli contamination thing of Huo Deing is not higher than 0.0005% according to the method described above, and purity can reach more than 99%, and specific activity can reach 1 * 10 ⁸International unit/more than the mg albumen.

Embodiment

Rely on embodiment to describe the present invention below, yet embodiment only illustrate, hard-core purpose.

Embodiment 1: fermentation

The following step relates to fermentation and induces the method for recombinant interleukin 2 analogue.

Material:

Substratum is by following the composition (every 1L):

Peptone 12g

Yeast extract 24g

Glycerine 4ml

KH ₂PO ₄ 2.31g

K ₂HPO ₄ 12.54g

50％MgSO ₄.7H ₂O 4ml

Purified water adds in right amount to 1L.

Coli strain is DH5 α pETIFN.

Inoculation:

Getting the cryodesiccated DH5 α pETIFN of a pipe is suspended in the above-mentioned substratum of 1ml.Suspension coating LB flat board, 37 ℃ of cultivations were inoculated in the above-mentioned substratum that contains 50 μ g/ml penbritins after 35-50 hour, put shaking table 100-300 rev/min, 37 ℃ and cultivated 5-10 hour, were extended to the requirement of inoculation fermentation jar again.

Under following condition, implement fermentation:

Substratum: above-mentioned substratum+50 μ g/ml penbritins+10 μ l/L defoamers

Temperature: 37 ℃

Dissolved oxygen: 50%

pH：6.4-7.4

Induce:

Under following condition, implement to induce:

OD600: greater than 15

Inductor: 1mM IPTG

Induction time: 3-6 hour

Identical between pH value, temperature and dissolved oxygen and yeast phase between inductive phase.

Induce and finish the centrifugal 20min collection of back 3000g thalline.

Embodiment 2: extract and the purifying inclusion body

Material:

Lysate: 0.05mol/L Tris.HCl-0.001mol/L EDTA-0.001mol/L DTT (pH7.5)

Denaturing soln: 0.05mol/L Tris.HCl-8mol/L urea-0.001mol/L DTT (pH7.5)

The supersound process lysing cell.Add lysate in the cell suspending liquid after cracking.Low temperature (2-10 ℃) is (14000rpm) centrifugal 30 minutes at a high speed, discards supernatant liquor, and precipitation suspends with lysate.Repeated centrifugation and suspension step 3 time.The inclusion body that obtains dissolves with denaturing soln.

Embodiment 3: the renaturation of inclusion body and purifying

Material and parameter:

Wavelength: 280nm

Chromatography media: Butyl Sepharose4 Fast Flow

The upper prop sample: dissolved inclusion body, volume are no more than 10 times of column volume

Flow velocity: 1cm/min

Buffer A: 0.1mol/L PBS-2mol/L (NH ₄) ₂SO ₄-8mol/L urea-0.001mol/L DTT (pH7.5)

Buffer B: 0.1mol/L PBS-2mol/L (NH ₄) ₂SO ₄-2mol/L urea-0.005mol/L Triptide (pH7.5)

Damping fluid C:0.1mol/L PBS-2mol/L urea (pH7.5)

The dissolved inclusion body metal chelate chromatography post of packing into.With buffer A washing and this chromatographic column of balance.Then the linear gradient with 15 times of column volumes carries out the transition to buffer B by buffer A, uses damping fluid C eluted protein at last.Collect elution peak by the absorbancy of measuring 280nm.

Embodiment 4: ion exchange chromatography

Material and parameter:

Wavelength: 280nm

Chromatography media: SP Sepharose

The upper prop sample: preceding step (embodiment 3) contains the elution peak of the recombinant interleukin 2 analogue of activity form

Flow velocity: 1cm/min

Buffer A: 0.02mol/L acetate buffer (pH3.0)

Buffer B: 0.02mol/L acetate buffer-0.15mol/L NaCl (pH3.0)

Collect on the elution peak of preceding step is packed ion exchange column into.With buffer A washing and balance chromatographic column, with buffer B wash-out target protein.Collect elution peak by the absorbancy of measuring 280nm.

Embodiment 5: gel exclusion chromatography

Material and parameter:

Wavelength: 280nm

Chromatography media: Sephcryl S-200

The upper prop sample: preceding step (embodiment 4) contains the elution peak of the recombinant interleukin 2 analogue of activity form

Flow velocity: 0.5cm/min

Damping fluid: 0.01mol/L PB-0.02%SDS (pH7.5)

Collect to regulate pH value to 7.5 to final concentration 0.02% and with NaOH, on the gel exclusion chromatography post of packing into from the elution peak adding SDS of preceding step solution.With wash-out target protein after damping fluid washing and the balance chromatographic column.Collect elution peak by the absorbancy of measuring 280nm.

Embodiment 6: pollutent, purity and specific activity are measured

Remaining e. coli protein is measured:

With Chinese People's Anti-Japanese Military and Political College's enterobacteria tropina antibody sandwich enzyme plate, sealing back adds sample and tropina standard substance, hatches 2 hours for 37 ℃, adds HRP enzyme connection Chinese People's Anti-Japanese Military and Political College enterobacteria tropina antibody after washing plate, hatched 1 hour for 37 ℃, add substrate after washing plate, hatched 40 minutes for 37 ℃, add sulfuric acid stop buffer termination reaction, enzyme plate is put into microplate reader, selecting the 492nm wavelength to detect, is the X value with standard substance concentration A, OD _492nmBe the Y value, among the input regression equation y=a+bx, calculate that the tropina residual quantity is 0.0003% in the interleukin II analogue sample of above-mentioned preparation.

Purity testing:

Preparation SDS-PAGE running gel, resolving gel concentration is 12.5%, concentrated gum concentration is 4.5%.Sample and sample buffer in 3: 1 ratio mixing after 100 ℃ water-bath 3-5 minute, in well, add sample 10 μ g.10mA begins electrophoresis with constant current, after tetrabromophenol sulfonphthalein dyestuff forward position enters separation gel electric current is transferred to 20mA, arrives the separation gel bottom until the tetrabromophenol sulfonphthalein dyestuff.Running gel carries out coomassie brilliant blue staining.Carry out scan process with scanner, the interleukin II analogue purity of above-mentioned preparation is 99.6%.

Specific activity is measured:

On 96 porocyte culture plates, every hole adds the interleukin II standard substance and the interleukin II analogue sample 50 μ l of 2 times of serial dilutions.Being made into cell concn after the CTLL-2 cell washs 3 times with the RPMI1640 nutrient solution is 6 * 10 ⁵The cell suspension of individual/ml is added on the 96 porocyte culture plates, every hole 50 μ l.Containing 5%CO ₂37 ℃ of incubators in cultivated 18-24 hour.Every hole adds 20 μ l MTT solution, continues containing 5%CO ₂37 ℃ of incubators in cultivated 4-6 hour.Every hole adds lysate 150 μ l, is containing 5%CO ₂37 ℃ of incubators in the insulation 18-24 hour.Liquid in the mixing cell plate is put into microplate reader, is reference wavelength with 630nm, measures absorbance in the 570nm place.The data computer program or four parametric regression computing methods are handled the calculation sample activity.Press the protein concentration of Lowry method working sample, the specific activity of calculation sample.The specific activity 1.2 * 10 of the recombinant interleukin 2 analogue of above-mentioned preparation ⁸IU/mg albumen.

Human interleukin-12 analogue aminoacid sequence table

1 Pro?Thr?Ser?Ser?Ser?Thr?Lys?Lys?Thr?Gln?Leu?Gln?Leu?Glu?His?Leu?Leu?Leu?Asp

21 Leu?Gln?Met?Ile?Leu?Asn?Gly?Ile?Asn?Asn?Tyr?Lys?Asn?Pro?Lys?Leu?Thr?Arg?Met?Leu

41 Thr?Phe?Lys?Phe?Tyr?Met?Pro?Lys?Lys?Ala?Thr?Glu?Leu?Lys?His?Leu?Gln?Cys?Leu?Glu

61 Glu?Glu?Leu?Lys?Pro?Leu?Glu?Glu?Val?Leu?Asn?Leu?Ala?Gln?Ser?Lys?Asn?Phe?His?Leu

81 Arg?Pro?Arg?Asp?Leu?Ile?Ser?Asn?Ile?Asn?Val?Ile?Val?Leu?Glu?Leu?Lys?Gly?Ser?Glu

101?Thr?Thr?Phe?Met?Cys?Glu?Thr?Ala?Asp?Glu?Thr?Ala?Thr?Ile?Val?Glu?Phe?Leu?Asn?Arg

121?Trp?Ile?Thr?Phe?Ser?Gln?Ser?Ile?Ile?Ser?Thr?Leu?Thr。

Claims (7)

1. One kind utilize escherichia coli expression and using hydrophobic chromatography purification and produce a kind of on aminoacid sequence with the similar proteic method of human interleukin-12, comprise the following steps: 1) Nucleotide of chemical synthesis coding interleukin II analogue, 2) intestinal bacteria that contain coding human interleukin-12 analogue nucleotide sequence are fermented, 3) extract inclusion body, 4) the using hydrophobic chromatography column carries out renaturation and purifying, it is characterized in that in the step (4) that the renaturation of inclusion body finishes and realize inclusion body purification simultaneously on the hydrophobic chromatography post, and the albumen specific activity behind the purifying is not less than 1 * 10 ⁸International unit/milligram albumen.
2. 2. according to claim 1, the albumen analogue has aminoacid sequence:

   Pro?Thr?Ser?Ser?Ser?Thr?Lys?Lys?Thr?Gln?Leu?Gln?Leu

   Glu?His?Leu?Leu?Leu?Asp?Leu?Gln?Met?Ile?Leu?Asn?Gly?Ile?Asn

   Asn?Tyr?Lys?Asn?Pro?Lys?Leu?Thr?Arg?Met?Leu?Thr?Phe?Lys?Phe

   Tyr?Met?Pro?Lys?Lys?Ala?Thr?Glu?Leu?Lys?His?Leu?Gln?Cys?Leu

   Glu?Glu?Glu?Leu?Lys?Pro?Leu?Glu?Glu?Val?Leu?Asn?Leu?Ala?Gln

   Ser?Lys?Asn?Phe?His?Leu?Arg?Pro?Arg?Asp?Leu?Ile?Ser?Asn?Ile

   Asn?Val?Ile?Val?Leu?Glu?Leu?Lys?Gly?Ser?Glu?Thr?Thr?Phe?Met

   Cys?Glu?Tyr?Ala?Asp?Glu?Thr?Ala?Thr?Ile?Val?Glu?Phe?Leu?Asn

   Arg?Trp?Ile?Thr?Phe?Ser?Gln?Ser?Ile?Ile?Ser?Thr?Leu?Thr
3. 3. according to claim 1, the albumen analogue is at expression in escherichia coli.
4. 4. according to claim 1, the albumen analogue of escherichia coli expression carries out renaturation and purifying with hydrophobic chromatography.
5. 5. according to claim 1,4 method, wherein said condition of carrying out renaturation on the hydrophobic chromatography post is to reduce the concentration of denaturing agent and add suitable oxygenant.
6. 6. according to claim 1,4,5 method, wherein said denaturing agent is a urea, can also be Guanidinium hydrochloride, sodium lauryl sulphate, and oxygenant wherein is a Triptide.
7. 7. according to claim 1, the specific activity of the albumen analogue behind the purifying is not less than 1 * 10 ⁸International unit/milligram albumen.