CN1280983A - Derivant of interleukin-11 and its preparing process - Google Patents

Abstract

A recombined interleukin-11 and its preparing process are disclosed. The CDNA of said IL-11 is obtained from that of wild-type IL-11 by losing 1-8 amino acids at N terminal or replacing them by other amino acids. The free IL-11 can be directly expressed and generated after the modified CDNA is put on expression carrier and transfered to colibacillus. The high-purity high-specific-activity recombined IL-11 can be prepared through collecting bacteria, breaking, solid-liquid separation, molecular sieve chromatography and ion exchange chromatography, and can be used to treat thrombocytopenia. Its advantages include direct expression of free IL-11 with expression level of 20% or more, simple purifying process, little heterologous protein pollution, and low cost.

Description

Derivative of interleukin 11 and preparation method thereof

The invention belongs to the recombinant protein drug in the field of medicaments.

Interleukin-11 (Interleukin11 is called for short IL-11) is a class important cytokine of human endocrine, and it acts on primary hemopoietic stem cell in the medullary cell, promotes megakaryocytic maturation, and the platelet increasing number promotes blood platelet regenerating.Recombinant interleukin 11 was gone on the market by drugs approved by FDA in November, 1997, became the treatment cancer patient through unique medicine of putting, platelet count reduces after the chemotherapy.

At present, producing IL-11 with gene engineering method is unique valid approach.But reorganization wild-type IL-11 runs into very big difficulty when expression in escherichia coli, promptly the IL-11 that free is complete can not be directly at expression in escherichia coli.This is because complete IL-11cDNA5 ' end GC content is abundant especially, in especially preceding 8 amino acid 6 proline(Pro) is arranged, and has seriously hindered complete IL-11 and has directly expressed.In order to express IL-11, can only use expression method every hop protein.So the difficulty that has caused fusion rotein to need enzyme to cut has again produced such as cutting with problems such as single-minded protease price costliness and heterologous protein pollutions.

The objective of the invention is directly to express free IL-11 with escherichia expression system the most frequently used in the genetically engineered.Under the constant prerequisite of the biologic activity that guarantees IL-11, invent out have the expression amount height, purifying process is simple, the novel recombinant interleukin 11 of low production cost and preparation method thereof.

Principle of the present invention: find in our test,, thereby reduce GC content and proline(Pro) number, the expression that can improve IL-11 at a part of amino acid of wild-type IL-11cDNA5 ' end disappearance.The amino acid number that lacks when IL-11N end is when 1 increases to 8, and the expression amount of free IL-11 grows out of nothing and progressively increases, and the biologic activity of expression product is compared then constant substantially with wild-type IL-11.Equally, if with proline(Pro) or glycine in Threonine and preceding 8 amino acid of methionine(Met) replacement IL-11, expression effect is identical with disappearance amino acid.Like this, just, can reach by disappearance N end several amino acid or with Threonine substituted prolines and glycine and in intestinal bacteria, directly express free IL-11.

Through the production bacterial classification that above-mentioned transformation transforms, after fermentation expression finished,----solid-liquid separation--sieve chromatography--purification steps such as ion exchange chromatography can make purity greater than 95% to bacterial cell disruption, and specific activity reaches 8 * 10 through receiving bacterium ⁶IU/mg has a bioactive novel recombinant interleukin 11 of the wild-type of being same as IL-11.

Most preferred embodiment: in the clone's process that makes up novel reorganization IL-11, its N end lacks preceding 5 amino acid (Pro-Gly-Pro-Pro-Pro) than the IL-11 of wild-type, and 6-8 amino acid changes Met-Thr-Thr into, and remaining aminoacid sequence is identical with the IL-11 of wild-type.The IL-11cDNA of above-mentioned transformation is placed among the expression vector PBV220, is transformed into bacillus coli DH 5 alpha, can directly express producing free IL-11 with temperature control inductive mode, and expression level reaches 20%.The free IL-11 that expresses is present in the colibacillary cytoplasm with solvable state, and tool natural radioactivity, this advantage can be removed the required problems such as sex change, renaturation and isomer separation of albumen of inclusion body form from, thereby has simplified purifying process greatly and reduced the production cost of IL-11.

Expression and preparation method are as follows: picking one single bacterium colony on the bacterial classification flat board, be inoculated in that (LBA: every liter contains the 10g peptone in the 2mlLBA substratum, the 5g yeast extract paste, 5gNaCl, the 60mg penbritin, PH7.0) 30 ℃ of overnight incubation are as seed, and the inoculum size by 1% is inoculated among the 50mlLBA, and 30 ℃ grow to 0D ₆₀₀, be warming up to 42 ℃ of abduction deliverings rapidly 4 hours, centrifugal results thalline at=0.5 o'clock.Thalline is suspended in 50mMPB (Na ₂HPO ₄+ NaH ₂PO ₄, PH8.0) in the damping fluid, the ultrasonication thalline, 20000g * 20 are minute centrifugal, get supernatant.Supernatant is crossed molecular sieve SephacrylS-200 gel chromatography, and applied sample amount is the 1-5% of column volume, and 20mMPB, PH8.0 buffer solution elution are collected IL-11 enrichment peak.Be further purified with cationic exchange coloum CM-Sepharose again,, wash the active peak of IL-11 at concentration place, the 0.25MNaCl left and right sides with 0-1MNaCl, 20mMPB, PH8.0 linear gradient elution.The SDS-PAGE electrophoretic analysis shows that the IL-11 purity that obtains is greater than 95%, and specific activity reaches 8 * 10 ⁶IU/mg.By aforesaid method, can obtain the novel reorganization IL-11 of high purity, high specific acitivity.

The invention provides a kind of new expression and prepare the method for IL-11, compare with U.S.'s Genetics Institute production technique of unique production IL-11 in the world and to have following characteristics: 1. expression level height, free IL-11 expression level of the present invention reaches 20%, and the IL-11 expression level of GI Company Inc. only is equivalent to 5%; 2. do not need cutting to use single-minded proteolytic enzyme, and the technology of GI Company Inc. need to come cleavage of fusion proteins with expensive enterokinase; 3. few heterologous protein pollutes, and exists the heterologous protein pollution problem of fusion rotein leading peptide and adding eterokinase in the technology of GI Company Inc.; 4. production technique is simple and with low cost.Above-mentioned advantage makes the present invention have tangible technological innovation and technology advance, make it more appropriate to suitability for industrialized production and economical and practical.

Claims (5)

1. the derivative of interleukin 11 is characterized in that the cDNA of this IL-11 holds 1-8 amino acid by other aminoacid replacement at the N of the IL-11 of wild-type cDNA 1-8 amino acid of end disappearance or N.

2. the preparation technology of the derivative of interleukin 11 is characterized in that through Escherichia coli fermentation--temperature-induced expression------solid-liquid separation--sieve chromatography--is finished bacterial cell disruption to receive bacterium by steps such as ion exchange chromatography.

3. solid-liquid separation as claimed in claim 2 is characterized in that separating with centrifugal method, and processing parameter is 15000g～25000g, and centrifugation time is 10～30 minutes.

4. sieve chromatography as claimed in claim 2 is characterized in that adopting S-100 or S-200, S-300 Sephacryl gel filtration chromatography, and applied sample amount is the 1-5% of column volume, and elution buffer is 10～30mMPB (PB:Na ₂HPO ₄+ NaH ₂PO ₄, PH6～9).

5. ion exchange chromatography as claimed in claim 2 is characterized in that adopting the CM-Sepharose cationic exchange coloum, and elution requirement is 0-1MNaCl, PH7～10, and linear gradient elution, best wash-out concentration is NaCl200～300mM.