CN1207305C - Preparing process and application of recombined human interleukin-11 - Google Patents

Abstract

The present invention relates to a method for preparing recombinant human interleukin-11 (IL-11). The present invention uses a principle that a genetic code has combination performance for designing a preference codon of colibacillus, a human IL-11cDNA segment amplified by PCR and a prokaryotic expression carrier such as pBV220, are assembled into a plasmid pIL-11 with high-efficiency expression, IL-11 gene expression is induced by a temperature after colibacillus is converted, and expression quantity reaches 20% of the total quantity of the protein of colibacillus bodies. Engineering bacteria are amplified by fermentation, an inclusion body is extracted after the engineering bacteria are crushed, the inclusion body is dissolved in urea by purification so as to be denatured, and a product is purified by a two-step method of gel exclusion chromatography and cation-exchange chromatography; molecule renaturation is carried out between two steps, and a freeze-dried pharmaceutical preparation for pharmaceutical use is prepared after a protective agent is added. The preparation can treats thrombocytopenia caused by reasons such as cancer chemotherapy, etc. and can reduce complicating symptoms such as bleeding, etc.

Description

A kind of preparation method of recombination human interleukins-11

The invention belongs to biological technical field, the medicine of the thrombocytopenia that the preparation that relates to human interleukin-11 itself or contain IL-11 causes as the treatment cancer chemotherapy.

Interleukin 11 is that support that nineteen ninety S.R.Paul etc. rebuilds hemopoietic stem cell in external long-term cultivation system at the research marrow stromal cell is done the time spent and found and isolated a kind of new cytokine, and this factor is in the external propagation that can stimulate the mouse plasmoma clone T1165 that IL-6 relies on.IL-11 has multiple effect to the marrow hemopoiesis tissue, IL-11 and some other can stimulate the pluripotency and the propagation of committed progenitor in different culture systems of primordial stem cell and different sources in the cytokine synergy that the cell proliferation and differentiation different steps is successively had an effect; IL-11 uses separately or is collaborative with other cytokine, and all there is hormesis in erythropoietic a plurality of stages; The differentiation and maturation of medullary system progenitor cell and the growth of bone-marrow-derived lymphocyte also there is promoter action; But its remarkable influence the most to hemopoietic tissue is to promote megalokaryocyte and hematoblastic generation, and this effect all is confirmed in the influence that comprises mouse, inhuman primate and people comprises external, in vivo test.Discover that further IL-11 also has significant biological effect to many non-hemopoietic tissues, all detects expression and the activity of IL-11 in brain, notochord neurone, enteron aisle and testis tissue.

Use separately in the interleukin-11 body and in mouse, rat, dog and primate, all show specific biological effect.In normal mouse, mainly act on the megakaryocytic series cell, can increase in the quantity, stimulating megakaryocyte born of the same parents of megalokaryocyte progenitor cell and duplicate and dose-dependently ground rising peripheral blood platelet number.Also show similar activity in non-human primate, administration animal peripheral blood platelet number raises and reaches 300%.In the severe bone marrow depression model and bone marrow transplant model that several chemotherapy, radiotherapy cause, use rhIL-11 can stimulate the polyphyly hemoposieis.In these models, all observe platelet recovery and accelerate, also observe the enhancing that neutrophil leucocyte and red corpuscle recover in the some of them model.These preclinical studies result has reconfirmed the extensive biologic activity that IL-11 showed in the experiment in vitro, and prompting IL-11 may be bone marrow depression and a thrombocytopenic medicine after a kind of effective treatment cancer chemotherapy and the bone marrow transplantation.

External thinking is the form by fusion rotein, one has or non-functional albumen or polypeptide on the N-terminal band, improve the proteic expression amount of hybridization, the excision of additional albumen or polypeptide can obtain target protein IL-11 (the Du et al:Blood of native sequences before utilizing special proteolytic enzyme with N-terminal again, 89:3897-3908,1997).The rhIL-11 that U.S. Genetic Institute produces promptly adopts above-mentioned thinking design, its expression product is a kind of fusion rotein of thioredoxin, need could obtain finished product through enterokinin enzyme cutting, greatest problem is a palpus with a large amount of expensive enterokinin enzymes.

What we adopted is the first kind of thinking that changes 5 ' terminal nucleotide sequence, has obtained good effect through enforcement.A free defective of expressing is that the N end often has unnecessary methionine(Met); and N-terminal Met can be excised by the methionine aminopeptidase in the E.coli; (Bio Essays such as Sherman; 1985:3; 27-31) once be reported in protokaryon and the eukaryotic cell; expressing protein if the rotation radius of the first amino acids residue when 1.22 dusts or littler (as Gly or Ala), then N-terminal Met can effectively be excised by methioninase.Initial with Gly.Because N-terminal do not contain methionine(Met), thereby IL-11 is heavy dose of can not cause producing antibody in the body when using, and affects the treatment.

The present invention adopts following technical proposals:

(1) property of merger and intestinal bacteria are arranged to the preference of amino acid code and the difference of human body cell in view of genetic code, specialized designs of the present invention has been synthesized people IL-11 gene 5 ' end and has been contained the dna fragmentation of intestinal bacteria preference codon, it can improve the expression level of IL-11 gene in intestinal bacteria on amino acid composition that does not change product people IL-11 and the basis that puts in order.Its step at first 5 ' and the guiding of 3 ' end primer under, go into certainly to amplify special people IL-11 cDNA fragment in the marrow stroma cell KW102 cell total rna.Pcr amplification product is inserted among the domestic efficient expression vector pBV220 that extensively adopts, be built into IL-11 expression plasmid pIL-11.It can be in intestinal bacteria HB101 and DH5 α high expression level, make IL-11 account for about 20% of bacterial protein, expression rate does not reduce through going down to posterity more than 100 times.

(2), as basic medium, suitably increase carbon source and nitrogenous source with ZYT by the fermentation engineering bacterium, by optimizing processing parameters such as dissolved oxygen, stirring velocity, fermentation pH value, final cultures OD600 value can reach about 20, and biomass is every liter of culture 20-25g, and the product expression rate is between 20-25%.The product expression amount can reach more than every liter of culture 200mg.

(3) set up purifying process on this basis, refining etc. comprising the fragmentation of thalline and inclusion body extracting, gel exclusion chromatography, renaturation and cation-exchange chromatography.

The process stabilizing of being set up, rate of recovery height, active good.This technology has following characteristics: 1. removed most of impurity by the washing of inclusion body, this is highly beneficial to later being further purified, and interleukin-11 content and concentration height in the extract help using the gel exclusion chromatography purifying in the first step; 2. the first step chromatography purification since in the extract interleukin-11 account for about 50% of total protein concentration, and impurity mostly is the albumen of molecular weight＞30kd greatly, purity of protein can reach more than 90% behind this step purifying; 3. renaturation behind the first step gel exclusion chromatography, renaturation concentration is 1mg/ml, keeps certain density NaCl or glycine in the renaturation, in case polymerization of interleukin-11 albumen and precipitation keep higher activity level simultaneously; 4. the final step ion exchange chromatography adopts the normal pressure filler, treatment capacity is big, the activity recovery height has been removed pyrogen and nucleic acid, phosphatide, lipopolysaccharides simultaneously, and the main points in this step are to add certain density glycine (concentration of glycine is 0.15M in our technology) in whole purge process.This technology to three batches of each 22.5L tunning purifying after every batch can get pure product 2.9-3.3g.

(4) after pure product add stablizer such as medicinal glycine and phosphate buffered saline buffer and solubility promoter, use the filtering with microporous membrane degerming, lyophilize powdering finished product.Use water for injection dissolving back, can be used for the treatment of the thrombocytopenia that reason such as cancer chemotherapy causes.

This product relies on strain 7TD-1 and B9-11 cell proliferation at external IL-11 and the IL-6 of stimulating, and can measure the biological activity of IL-11 in view of the above.

Rendeing a service the experimental study content comprises: the external influence that people's normal bone marrow hemopoietic progenitor cell colony is formed of rhIL-11; Subcutaneous injection is to the influence of normal Balb/C male mice peripheral blood platelet and the formation of hemopoietic progenitor cell colony; Right ⁶⁰The therapeutic action of thrombocytopenia due to Co full-body exposure and the carboplatin abdominal injection; Therapeutic action to macaque thrombocytopenia due to the carboplatin intravenous drip; Platelet function index and dynamic change thereof.

The result shows: subcutaneous injection rhIL-11 can alleviate the decline degree of the peripheral blood platelet number that irradiation and carboplatin injection caused significantly, promotes the recovery of platelet count.100-400 μ injection in g/kg/ days mouse, 50-100 μ injection in g/kg/ days monkey all has good curative effect, treat the 13-15 days platelet counts in back and return to the pre-irradiation level, obviously shift to an earlier date than model control group time of recovery, extended treatment can make platelet count surpass normal level, dosage strengthens time of recovery in advance, and the power that rhIL-11 renders a service is relevant with the thrombopenia degree, more remarkable treatment effect when thrombopenia is obvious; RhIL-11 also can obviously improve platelet count to normal mouse, external to people and mouse bone marrow cells grain system, red system, megakaryoblast forms that obvious effect is all arranged, but injection does not have obvious influence to peripheral blood leucocyte, red corpuscle in the body.The new platelet function that produces is normal.

The present invention uses recombinant DNA technology, under the prerequisite that does not change coded amino acid, changes gene 5 ' end parts Nucleotide, thus the expression that has improved product, and product exists with loose inclusion body form, and molecule is easy to the sex change renaturation.The downstream purification mode is easy, and the yield height reaches the specification of quality of clinical application.This product is used for the treatment of experimental thrombocytopenia, can obtain significant curative effect.

The present invention describes with following example:

Embodiment 1, PCR design of primers and people IL-11 gene amplification

People IL-11 protein molecule is made of 178 amino-acid residues, wherein N-terminal Pro disappearance back is initial with Gly, a N-terminal amino acid row are Gly-Pro-Pro-Pro-Gly-Pro-Pro-Arg, because protogene 5 ' terminal sequence GC content is too high, be unfavorable in prokaryotic system, expressing, therefore the primary thought of the design is to reduce by 5 ' end GC content, and 5 after the design ' end primer sequence is:

EcoRI

5′-CG? GAATTC?ATG?GGT?CCA?CCA?CCT?GGT?GGT?CCA?CCT

3 ' end primer sequence is:

SmaI

5′- CCC?GGG?TCA?CAG?CCG?AGT?CTT?CAG?CAG-3′

Adopt synthetic above-mentioned two oligodeoxynucleotides of solid phase tris phosphite method, the separation and purification of preparation gel electrophoresis.

Adopt micro-guanidinium isothiocyanate/phenol/chloroform single stage method total RNA of purifying in the matrix continuous cell line KW102 of people's derived from bone marrow cell, make A260/A280 ratio＞1.8, get the total RNA of 10 μ l (about 5 μ g), add 50pmol 3 ' end primer, 70 ℃ of thermally denature 5min, the room temperature cooling is lowered the temperature it naturally, after waiting to reduce to room temperature, adds following sample in 42 ℃ of water-baths successively:

Reverse transcription reaction system: 5 * reverse transcription damping fluid, 5 μ l

RNA enzyme inhibitors 20U

40nM trisodium phosphate 3 μ l

AMV reversed transcriptive enzyme 15U (1 μ l)

DNTP (each 2.5mM) 2 μ l

Add no RNA enzyme water to 25 μ l

42 ℃ of water-bath 60min of said mixture add H ₂70 ℃ of effects of O 75 μ l 5min deactivation ThermoScript II, this reactant can be used as the starting template of pcr amplification, and negate transcription product 20 μ l add following ingredients:

10 * PCR damping fluid, 10 μ l

DNTP (each 2.5mM) 16 μ l

5 ' and each 40pmol of 3 ' both sides primer

Add H ₂O to 99.5 μ l

95 ℃ of said mixtures, add 0.5 μ l Tap archaeal dna polymerase 2.5U behind the 2min, mixing, 61 ℃ of annealing of 95 ℃ of sex change, 72 ℃ of extensions each 60 seconds, carry out extending 8min behind 35 round-robin amplified reactions, get reactant and carry out electrophoresis and identify the single band observe 0.55kb, meet the length of complete people IL-11 gene, and can with 5 of mark ' end primer hybridization.

Embodiment 2, expression and the sequential analysis of people IL-11 gene in intestinal bacteria

With pcr amplification product and the expression vector plasmid pBV220 of restriction enzyme EcoRI and SmaI double digestion 0.55kb, separate through agarose gel electrophoresis, reclaim the dna fragmentation of 0.55kb and 3.66kd respectively.Mole such as two fragments mixes, and 12～16 ℃ of ligations of T4 dna ligase are spent the night, and connector is transformed in the E.coli bacillus coli DH 5 alpha of CaCl2 processing, through the penbritin screening and culturing.Select single bacterium colony, the preparation plasmid is identified with digestion with restriction enzyme, contains the correct person of people IL-11 gene fragment and direction of insertion, called after pIL-11, corresponding engineering bacterium called after pIL-11/DH5 α.

The engineering bacteria that contains above expression plasmid has more a protein band through amplification cultivation, temperature-induced about 19kd.This albumen can manifest specific reaction with people IL-11 monoclonal antibody.

Under the sequencing primer guiding, survey from two respectively the people IL-11 gene fragment of inserting is carried out dna sequence analysis, affirmed that institute's amplification PCR products meets Design Conception, its amino acid sequence coded identical with people IL-11 sequence (nucleotide sequence and the amino acid sequence corresponding that record see Table 1).

The fermentative production of embodiment 3, people IL-11

After 30 ℃ of incubated overnight of engineering bacteria, be inoculated in the 2YT substratum in 5% ratio, 30 ℃ are cultivated OD600 is 2.0 o'clock, and temperature is risen to 42 ℃, and this moment, IL-11 promptly induced generation, collected thalline behind the 4h.Add nutrient substances such as nitrogenous source, carbon source on the different opportunitys of expressing.Final tunning density can reach 20, and biomass is every liter of culture 20-25g.Electrophoresis is identified the IL-11 expression level, thin layer scanning show IL-11 account for bacterial protein 20% between.

The preservation of embodiment 4, engineering bacteria and stability

Engineering bacteria adds 30% glycerine ,-20 ℃ of preservations.Short-term is preserved bacterial classification can use soft agar LB flat board.Be to check the stability of engineering bacteria, with original strain, 50 generations and 100 generation bacterium respectively the extracting plasmid carry out enzyme and cut evaluation, the three is identical as a result.The product expression level of three bacterium amplification cultivation things is suitable simultaneously, proves that the engineering bacteria among the present invention is very stable.

The separation and purification of embodiment 5, IL-11

People IL-11 exists with loose inclusion body form in thalline, recovery, purifying inclusion body behind the bacterial cell disruption, supernatant at first passes through the gel exclusion chromatography preliminary purification behind the 5M urea extraction, and IL-11 is refining through cation-exchange chromatography again after the renaturation, can obtain the pure product of IL-11 more quickly.

Gel exclusion chromatography post Sephacryl S-200 is through 50mM PB, pH7.0,1mM EDTA-Na ₂, go up sample after the 5M urea balance, substep is collected the IL-11 of 19kd behind the last sample, IL-11 behind the preliminary purification removes urea with the substep dialysis method, finish renaturation process, sample is further refining through CM-Sepharase Fast Flow again after the renaturation, and collecting molecular weight is the pure product of IL-11 of 19kd.

The evaluation of embodiment 6, IL-11 product

SDS-PAGE and HPLC purity checking are all more than 98%, and 7TD1 and B9-11 cell strain mtt assay are measured its biological activity, and specific activity is 8 * 10 ⁶More than the U/mg.N-terminal protein sequence analysis result is except that N end disappearance Pro, and all the other are identical with native protein in the body, and order is G.P.P.P.G.P.P.R.V.S.P.D.P.R.A.

Embodiment 7, sterile filtration, packing, freeze-drying

According to protein content and activity, add auxiliary material and stablizer in following ratio

Every of rhIL-11 contains 0.75,1.5 and 3.0mg, and buffer system is 10mM PB, pH7.0, and the above auxiliary material of 0.3M glycine must meet the requirement of injecting drug use.

Sterile filtration: reach at cleanliness factor under 100 grades the condition, with 0.22 μ M filtering with microporous membrane, packing, freeze-drying, seal, labeling, vanning.Be stored in 2～8 ℃ the cold storage environment.

1 GGT?CCA?CCA?CCT?GGT?CCA?CCT?CGA?GTT?TCC?CCA?GAC?CCT?CGG?GCC?GAG?CTG?GAC

2 Gly?Pro?Pro?Pro?Gly?Pro?Pro?Arg?Val?Ser?Pro?Asp?Pro?Ary?Ala?Glu?Leu?Asp

1 10

1 AGC?ACC?GTG?CTC?CTG?ACC?CGC?TCT?CTC?CTG?GCG?GAC?ACG?CGG?CAG?CTG?GCT?GCA?CAG?CTG

2 Ser?Thr?Val?Leu?Leu?Thr?Arg?Ser?Leu?Leu?Ala?Asp?Thr?Arg?Gln?Leu?Ala?Ala?Gln?Leu

20 30

1 AGG?GAC?AAA?TTC?CCA?GCT?GAC?GGG?GAC?CAC?AAC?CTG?GAT?TCC?CTG?CCC?ACC?CTG?GCC?ATG

2 Arg?Asp?Lys?Phe?Pro?Ala?Asp?Gly?Asp?His?Asn?Leu?Asp?Ser?Leu?Pro?Thr?Leu?Ala?Met

40 50

1 AGT?GCG?GGG?GCA?CTG?GGA?GCT?CTA?CAG?CTC?CCA?GGT?GTG?CTG?ACA?AGG?CTG?CGA?GCG?GAC

2 Ser?Ala?Gly?Ala?Leu?Gly?Ala?Leu?Gln?Leu?Pro?Gly?Val?Leu?Thr?Arg?Leu?Arg?Ala?Asp

60 70

1 CTA?CTG?TCC?TAC?CTG?CGG?CAC?GTG?CAG?TGG?CTG?CGC?CGG?GCA?GGT?GGC?TCT?TCC?CTG?AAG

2 Leu?Leu?Ser?Tyr?Leu?Arg?His?Val?Gln?Trp?Leu?Arg?Arg?Ala?Gly?Gly?Ser?Ser?Leu?Lys

80 90

1 ACC?CTG?GAG?CCC?GAG?CTG?GGC?ACC?CTG?CAG?GCC?CGA?CTG?GAC?CGG?CTG?CTG?CGC?CGG?CTG

2 Thr?Leu?Glu?Pro?Glu?Leu?Gly?Thr?Leu?Gln?Ala?Arg?Leu?Asp?Arg?Leu?Leu?Arg?Arg?Leu

100 110

1 CAG?CTC?CTG?ATG?TCC?CGC?CTG?GCC?CTG?CCC?CAG?CCA?CCC?CCG?GAC?CCG?CCG?GCG?CCC?CCG

2 Gln?Leu?Leu?Met?Ser?Arg?Leu?Ala?Leu?Pro?Gln?Pro?Pro?Pro?Asp?Pro?Pro?Ala?Pro?Pro

120 130

1 CTG?GCG?CCC?CCC?TCC?TCA?GCC?TGG?GGG?GGC?ATC?AGG?GCC?GCC?CAC?GCC?ATC?CTG?GGG?GGG

2 Leu?Ala?Pro?Pro?Ser?Ser?Ala?Trp?Gly?Gly?Ile?Arg?Ala?Ala?His?Ala?Ile?Leu?Gly?Gly

140 150

1 CTG?CAC?CTG?ACA?CTT?GAC?TGG?GCC?GTG?AGG?GGA?CTG?CTG?CTG?CTG?AAG?ACT?CGG?CTG?TGA

2 Leu?His?Leu?Thr?Leu?Asp?Trp?Ala?Val?Arg?Gly?Leu?Leu?Leu?Leu?Lys?Thr?Arg?Leu

160 170

Table 1.IL-11 cDNA and aminoacid sequence

(1. nucleotide sequence 2. aminoacid sequences)

Claims (1)

1, the preparation method of a kind of recombination human interleukins-11 (IL-11) comprises the gene of PCR design of primers, amplification, the structure of expression plasmid, the purifying process of expression product, it is characterized in that:

(1) 5 ' end PCR primer and amplification cDNA 5 ' terminal sequence are:

5′-CG? GAATTC?ATG?GGT?CCA?CCA?CCT?GGT?CCA?CCT

Wherein the underscore sequence is the EcoRI site,

(2) above-mentioned IL-11 cDNA is assembled into efficient expression plasmid pIL-11 with the prokaryotic expression carrier pBV220 that contains PL, PR promotor;

(3) pIL-11 transformed into escherichia coli, with temperature-induced IL-11 expression of gene, product exists in the engineering bacteria with the inclusion body form;

(4) by fermentation technique amplification engineering bacteria, make basic medium, add nutrient substances such as nitrogenous source, carbon source different opportunitys expressing with 2YT;

(5) the broken thalline in fermentation back, the purifying inclusion body also dissolves sex change with urea, adopts gel exclusion chromatography and cation-exchange chromatography two step method purified product subsequently, carries out the molecule renaturation between two steps.