CN1167712C - Interleukin 11 analog - Google Patents
Interleukin 11 analog Download PDFInfo
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- CN1167712C CN1167712C CNB011100818A CN01110081A CN1167712C CN 1167712 C CN1167712 C CN 1167712C CN B011100818 A CNB011100818 A CN B011100818A CN 01110081 A CN01110081 A CN 01110081A CN 1167712 C CN1167712 C CN 1167712C
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- 102000003815 Interleukin-11 Human genes 0.000 title abstract description 37
- 108090000177 Interleukin-11 Proteins 0.000 title abstract description 37
- 229940074383 interleukin-11 Drugs 0.000 title abstract description 32
- 101001010568 Homo sapiens Interleukin-11 Proteins 0.000 claims abstract description 8
- 102000049885 human IL11 Human genes 0.000 claims abstract description 7
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical class [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
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- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides interleukin 11 which relates to an important cell factor and has the main function for stimulating bone marrow stem cells to carry out cell differentiation towards a macronucleus system; therefore, the enlargement and the maturation of megakaryocyte are promoted, and the content of blood platelets of peripheral blood is improved. At present, the recombinant human interleukin 11 of genetic engineering is already on the market in US, and the main clinic indication is the supportive treatment of blood platelet reduction after radiotherapy; the present invention has more obvious and exact effect than thrombopoietin, interleukin 6, etc., and a medication patient has no adverse reaction; clinical research and test are carried out in China, and the present invention can be on the market at the end of 2002. The present invention improves the weakness that natural IL-11 is easily in acidolysis and strengthens the stability in vitro by the reorganization of IL-11 protein.
Description
Interleukin-11 main effect in vivo is to promote that macronucleus is hematopoiesis, improves the platelet content of peripheral blood, simultaneously with the collaborative immunity of organism of regulating of other cytokine.In U.S.'s listing, commodity are called " neumega " to human interleukin 11 at present, and clinical indication is a thrombopenia supportive treatment behind the tumour patient chemicotherapy, patient's peripheral blood platelet counts can be improved 2-3 doubly, obviously improve coagulation function.Animal experiment shows that interleukin-11 reaches about 1.5 times the peripheral blood PC raising of normal mouse and macaque, and no obvious toxic-side effects is found.
Because interleukin-11 is to be that the form that cell passes through paracrine, autocrine is regulated hematoblastic generation by marrow stromal cell and macronucleus mainly in the body, participating in cytokine network simultaneously regulates the far-end of immunologic function and still relies on monokaryon-the engulf paracrine of system, making body must set up some degradeds or deactivation system is the regulation and control of hematopoiesis and immunity function to be easy in time to carry out necessary macronucleus, so the transformation period of natural interleukin 11 in marrow and peripheral blood is shorter.Recent discovers; influence the unstable that major reason is its protein amino acid sequence of transformation period in the interleukin-11 body; except that by the proteasome degradation of body; easily being hydrolyzed (especially acidolysis) also is a kind of principal character; normal or the not compensatory thrombopenia patient for the hemopoietic system function, it is necessary for improving the peripheral blood platelet content to prolong in the body of interleukin-11 the transformation period.As the medicine that applies to human body, overcome interleukin-11 easily by the weakness of acidolysis, have and improve drug effect, reduce patient's usage quantity, extend the expiration date, reduce significant social and economic benefit such as cost.
The external degradation of interleukin-11 is mainly caused by acid catalysis.Under the faintly acid effect, peptide bond rupture between the 134th Asp of natural human interleukin-11 peptide chain and the 135th Pro, sequencing result and electrophoresis result are all supported this conclusion.This patent has solved this problem effectively by natural human interleukin-11 the 134th amino acids Asp is sported Asn, and the capacity antacid of interleukin-11 is obviously strengthened, and is difficult for by the acidolysis inactivation in the weak acid injection that human body can tolerate.The 134th amino acids transformation mainly contains following reason to this patent to interleukin-11: the mechanism that (one) interleukin-11 is ruptured by acid catalysis infers it is (COOH) to interact with the pyridine ring of the 135th Pro and cause because the carboxyl of the 134th Asp, the carboxyl of Asp is replaced with the amide group of Asn, can solve the problem of the fracture of peptide bond acidolysis herein preferably.(2) by reporting to the simulation of interleukin-11 secondary structure and according to data of literatures, find that the 134th of natural interleukin 11 and the 135th amino acids are in the random coil zone, therefore suddenly change herein, can not influence the formation of its secondary structure and tertiary structure, it is bioactive stable promptly to guarantee to transform the back interleukin-11.(3) sport Asn by Asp, do not influence original carbochain skeleton structure of interleukin-11 and quantity, less to its biological activity, immunogenicity and metabolic way change in vivo, chance appears thereby reduce extra toxic side effect.
In addition, 10 amino acid artifact activity of the N-terminal of natural human interleukin-11 disappearance are constant.By the forecast analysis (Anthewin software) to the interleukin-11 secondary structure, 10 amino acid of N-terminal are random coil.Because most of zone of natural interleukin 11 belongs to imporosity, link together by hydrophobic interaction, and the open structure of its N-terminal is exposed in the extramolecular solution, stability weakens relatively, do not influence the biological activity of IL-11 simultaneously behind 10 amino acid of N-terminal disappearance, so this patent at first lacks 10 amino acid of N-terminal by molecular designing.The 10th amino acids Ala cuts the back by enzyme and keeps.By above transformation, 9 amino acid of N-terminal disappearance have finally been obtained, 1st, 125 (be equivalent to natural IL-11 the 10th, 134) amino acid are respectively the interleukin 11 analog of Ala, Asn, and this analogue has stronger antiacid ability of separating and stability simultaneously in that water-soluble and biological activity is impregnable.
Example:
1. the acquisition of the structure of interleukin 11 analog plasmid and engineering bacteria
The template and the design of primers that go out interleukin 11 analog by pcr amplification are as follows:
Seq 1. templates: 5 ' GGG GCA CTG GGA GCT CTA CAG CTC CCA GGT GTG
CTG?ACA?AGG?CTG?CGA?GCG?GAC?CTA?CTG?TCC?TAC?CTG?CGG?CAC?GTG
CAG?TGG?CTG?CGC?CGG?GCA?GGT?GGC?TCT?TCC?CTG?AAG?ACC?CTG?GAG
CCC?GAG?CTG?GGC?ACC?CTG?CAG?GCC?CGA?CTG?GAC?CGG?CTG?CTG?CGC
CGG?CTG?CAG?CTC
Seq 2.5 ' primer sequence 1:ACG CGG CAG CTG GCT GCA CAG CTG AGG
GAC?AAA?TTC?CCA?GCT?GAC?GGG?GAC?CAC?AAC?CTG?GAT?TCC?CTG?CCC
ACC?CTG?GCC?ATG?AGT?GCG?GGG?GCA?CTG?GGA?GCT?CTA?CAG?CTC
Seq 3.5 ' primer sequence 2:TCC TCT AGA GCT TCC CCA GAC CCT CGG GCC
GAG?CTG?GAC?AGC?ACC?GTG?CTC?CTG?ACC?CGC?TCT?CTC?CTG?GCG?GAC
ACG?CGG?CAG?CTG?GCT?GCA?CAG
Seq 4.3 ' primer sequence 1:CCT GAT GCC CCC CCA GGC TGA GGA GGG GGG CGC
CAG?CGG?GGG?CGC?CGG?CGG?GTT?CGG?GGG?TGG?CTG?GGG?CAG?GGC?CAG?GCG
GGA?CAT?CAG?GAG?CTG?CAG?CCG?GCG?CAG?CAG?CCG
Seq 5.3 ' primer sequence 2:ATT GAA TTC TTA TCA CAG CCG AGT CTT
CAG?CAG?CAG?TAG?CCC?CCT?CAC?GGC?CCA?GTC?AAG?TGT?CAG?GTG?CAG
CCC?CCC?CAG?GAT?GGC?GTG?GGC?GGC?CCT?GAT?GCC?CCC?CCA?GGC?TGA
GGA
Above Seq3 and Seq4 primer and Seq1 single-stranded template are carried out the product about pcr amplification acquisition 350bp, be template with this product again, carry out pcr amplification with Seq2 and Seq5 primer, end product is checked through 5% sex change PAGE electrophoresis, obtains containing about a 550bp interleukin 11 analog gene fragment of corresponding protein restriction enzyme site.
Purpose fragment and plasmid pGEX-4T-1 reclaim big fragment respectively behind BamHI and EcoRI double digestion, connect recombinant plasmid called after pGEX-IL11a (Fig. 1) with the T4 ligase enzyme at 18 ℃.Use CaCl then
2Method transformed into escherichia coli JM109 is coated on and contains 50ug/ml penbritin LA flat board, selects positive bacteria and drops on and be cultured to OD among the LA
600Add IPTG 1.0mM during for 0.6-0.8 and induce 3-4 hour centrifugal collection thalline, occur the protein band about a 18KD behind the broken bacterium of 8M urea during the 10%SDS-PAGE electrophoresis, expression amount is 40%.Monoclonal antibody (DSL product, the U.S.) immunoblotting calibrating through natural type IL-11 shows positive reaction.The engineering bacteria called after IL-11a/JM109 that efficiently expresses IL-11a that is obtained.
2. fermentation
The IL-11a/JM109 that contains the interleukin 11 analog gene, in the LA substratum that contains 100 μ g/ml penbritins, shake bottle and spend the night (37 ℃, 200rpm), contain in the LA substratum of 100 μ g/ml penbritins by inoculation in 1: 30 again, cultivate after 3 hours for 37 ℃, add 0.5mM IPTG and induced 4 hours.Collect thalline through the SDS-PAGE electrophoretic analysis, the fusion rotein GST-IL-11a (44.6KD) that finds to contain interleukin 11 analog is based on solubility expression, and expression amount accounts for 25% of bacterial protein.
3. purifying
Reorganization I1-11a protein expression is finished by the IL-11a/JM109 engineering bacteria.The centrifugal collection thalline of fermentation secondary fermentation liquid, bacterium is broken in pressure homogenate, collect Glutathione Sepharose chromatography column on the supernatant, wash fusion rotein with the elutriant that contains reduced glutathion (GSH) after the balance, adding 37 ℃ of enzymes of zymoplasm (5NIHU/mL) cut 2 hours, remove GST albumen by cation-exchange chromatography (SP Sepharose Fast Flow) then, obtain purity greater than 95% reorganization I1-11a.
4. biological activity assay
4.1 7TD1 cell strain method
Basis cell culture fluid: 1640 basic medium 10.0g, NaHCO
32.2g, Hepes5.9g, Sodium Propionate 0.11g, 0.05M mercaptoethanol 2.0ml, ddH
2O 1000ml; Cell culture medium: basic cell culture medium 90ml, calf serum 10ml, two anti-(penicillin+Streptomycin sulphate) 100U/ml, gsh 0.03g/ml; Survey the substratum of living: basic medium 95ml calf serum 5.0ml, two anti-glutathione concentrations that reach are with last identical; MTT solution: MTT powder 5.0mg, 0.01M phosphoric acid buffer 1.0ml, pH7.2 filtration sterilization; Lysate: SDS 10g,, DMSO 25ml, ddH
2O 19ml transfers PH=4.7.
Method: after the 7TD1 cell strain goes down to posterity, select the animated period cell to wash 3 times furnishing 1 * 10 with basic cell culture medium
5/ ml, testing sample dilutes to survey liquid alive, the above enchylema of adding in 96 orifice plates (after the dilution) 90 μ l/ holes, add each extent of dilution testing sample 10 μ l/ hole, under 37 ℃, 48-72 hour, add MTT, 10 μ l/ holes, continue to cultivate 4-6 hour, add lysate, 100 μ l/ holes, continue to cultivate 12 hours, read OD
570Calculated activity.
Table 1. experimental result:
Weaker concn (μ g/ml) 10
210
110
010
-110
-210
-310
-4
numega 0 0.025 0.040 0.025 0.022 0.015 0.01
Sample 0.018 0.021 30.04 0.029 0.022 0.009 0.002
The specific activity of interleukin analog is: 2.5 * 10
6Unit/mg.
4.2 the variation of injection interleukin 11 analog peripheral blood platelet counts after the mouse carboplatin chemotherapy
Kunming mouse intravenous injection carboplatin 40mg/kg/day for three days on end, injects 100ug/ time/day of subcutaneous injection interleukin 11 analog in back 18 hours, successive administration 14 days.
Table 2. interleukin 11 analog is to the influence of injection carboplatin mouse platelets counting
Time platelet count (10
9/ L)
(my god) model control group 100 μ KD organize neumega (100 μ KD)
Administration preceding 778.1 ± 178.1 754.2 ± 169.0 778.0 ± 132.1
Administration 3 days 769.2 ± 99.4 821.3 ± 164.1 810.3 ± 122.5
7 days 273.5 ± 51.0 1306.7 ± 179.5
*1256.3 ± 179.7
11 days 130.7 ± 31.7 1189.7 ± 189.8
*800.3 ± 125.2
*
13 days 399.6 ± 62.7 767.9 ± 141.8
* *583.7 ± 199.3
Drug withdrawal 3 days 867.9 ± 156.0 867.1 ± 69.3 707.0 ± 287.0
7 days 872.0 ± 156.3 980.0 ± 177.0 851.4 ± 191.0
11 days 914.2 ± 179.3 1169.5 ± 289.9
*1064.9 ± 277.7
*
Annotate: neumega is that recombination human interleukin 11 using and the model control group that the U.S. has gone on the market compares * P＜0.05, * * P＜0.01, * * * P＜0.001
As can be seen from Table 2, using interleukin 11 analog and neumega supportive treatment through the carboplatin chemotherapy mouse, the 7th day peripheral blood platelet counts of medication peaks, and falls after rise gradually afterwards to the 13rd day low ebb of medication; Drug withdrawal bottom out in the time of the 3rd day, drug withdrawal reached normal level on the 11st day.Yet the interleukin 11 analog test group is considerably slower than neumaga in medication peripheral blood platelet counts decline afterwards in the 7th day, and the recovery of peripheral blood platelet counts is significantly accelerated after the drug withdrawal.This possibility of result prolongs relevant with the interleukin 11 analog transformation period in vivo.
Fig. 1 is the design of graphics of pGEX-IL-11 plasmid.
Claims (2)
1. human interleukin 11 analogue, its aminoacid sequence is:
01-19：Ala?Ser?Pro?Asp?Pro?Arg?Ala?Gln?Leu?Asp?Ser?Thr?Val?Leu?Leu?Thr?Arg?Ser?Leu
20-37：Leu?Ala?Asp?Thr?Arg?Glu?Leu?Ala?Ala?Gln?Leu?Arg?Asp?Lys?Phe?Pro?Ala?Asp
38-56：Gly?Asp?His?Asn?Leu?Asp?Ser?Leu?Pro?Thr?Leu?Ala?Met?Ser?Ala?Gly?Ala?Leu?Gly
57-75：Ala?Leu?Gln?Leu?Pro?Gly?Val?Leu?Thr?Arg?Leu?Arg?Ala?Asp?Leu?Leu?Ser?Tyr?Leu
76-94：Arg?His?Val?Gln?Trp?Leu?Arg?Arg?Ala?Gly?Gly?Ser?Ser?Leu?Lys?Thr?Leu?Glu?Pro
95-112：Glu?Leu?Gly?Thr?Leu?Gln?Ala?Arg?Leu?Asp?Arg?Leu?Leu?Arg?Arg?Leu?Glu?Leu
113-131：Leu?Met?Ser?Arg?Leu?Ala?Leu?Pro?Gln?Pro?Pro?Pro?Asn?Pro?Pro?Ala?Pro?Pro?Leu
132-150：Ala?Pro?Pro?Ser?Ser?Ala?Try?Gly?Gly?Ile?Arg?Ala?Ala?His?Ala?Ile?Leu?Gly?Gly
151-168：Leu?His?Leu?Thr?Leu?Asp?Trp?Ala?Val?Arg?Gly?Leu?Leu?Leu?Leu?Lys?Thr?Arg
169：Leu
2. the human interleukin 11 analogue in the claim 1 uses intestinal bacteria to be host cell, and the method for using gene engineering is expressed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011100818A CN1167712C (en) | 2001-03-30 | 2001-03-30 | Interleukin 11 analog |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011100818A CN1167712C (en) | 2001-03-30 | 2001-03-30 | Interleukin 11 analog |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1377893A CN1377893A (en) | 2002-11-06 |
| CN1167712C true CN1167712C (en) | 2004-09-22 |
Family
ID=4658330
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011100818A Expired - Lifetime CN1167712C (en) | 2001-03-30 | 2001-03-30 | Interleukin 11 analog |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20070160577A1 (en) * | 2005-12-06 | 2007-07-12 | Wyeth | Interleukin-11 compositions and methods of use |
| CN114561395B (en) * | 2022-03-30 | 2023-07-28 | 四川大学 | Fusion tag-free rhIL-11 and soluble expression and efficient purification method of mutant thereof |
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