CN1197874C - Modification and preparation of human interleukin 11 - Google Patents
Modification and preparation of human interleukin 11 Download PDFInfo
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Abstract
The present invention relates to human interleukin 11 with the functions for stimulating megakaryocyte hyperplasia, increasing the quantity of blood platelets, adjusting immunological function, protecting mucosa, etc.; the recombination product can be applied to the treatment thrombocytopenia caused by chemotherapy for patients with tumors. The present invention adopts a genetic engineering method to prepare novel improved type recombination human interleukin 11 which is verified to have the same biological functions in vivo and in vitro with natural human interleukin 11, and can become a medicine for treating the thrombocytopenia.
Description
Verified and discovery plays regulating effect in immunity system and hemopoietic system protein has tens of kinds, and they are referred to as cytokine, as G CFS, interleukin-, Interferon, rabbit, tumour necrosis factor etc.These cytokines have biological function and target cell widely widely, comprise medullary cell, peripheral blood cells, fetal liver cell, lymphocyte etc.Because the natural source of cytokine is very limited, the using gene engineering means are carried out gene clone, recombinant expressed and purifying efficiently, are present numerous cytokine important means in fundamental research and clinical application.
(interleukin 11, IL-11) are the albumen with multiple biological function for human interleukin 11.The main effect that confirms has the stimulation hyperplasia megakaryocytic, promotes B cells whose development and function, and the protection mucomembranous cell is impaired, platelet increasing quantity.Natural ripe human interleukin 11 is made up of 178 amino acid, and molecular weight is 20kD, is strong basicity albumen (pI 11.7), and hydrophobicity is stronger.1100 pairs of bases of people IL-11 full length gene, wherein 5 ' end has 72 base non-coding regions, and 3 ' end has 431 base non-coding regions.Reading frame is 597 bases, accurate translation is the human interleukin 11 albumen that 199 amino acid are formed, wherein N-terminal 1-21 amino acid is signal peptide sequence, so the human interleukin 11 N-terminal of natural radioactivity is Pro Gly Pro Pro Pro Gly Pro Pro Arg Ala Ser ProAsp etc.The interleukin 11 gene of ape and mouse is by clone and recombinant expressed success.The interleukin 11 of people and ape is having high same preface aspect amino acid and the base sequence, wherein amino acid sequence homology 93.7%, base sequence homology 99.5%.
Recombination human interleukin 11 using is expressed successfully at prokaryotic cell prokaryocyte (intestinal bacteria) and eukaryotic cell (COS cell).Because the singularity of IL-11 is very difficult at expression in escherichia coli.U.S. Genetics Institute company adopts the expressing fusion protein pTRXFUS of system to come expressing human IL-11 to succeed, and its fusion rotein is a reduced form sulphur oxygen albumen (Thioredoxin).The pI that this system has several characteristics extremely to help expression (1) Thioredoxin of people IL-11 is 4.5, and the indirect Head Section has four aspartic acids, can in and the strong basicity of IL-11; (2) Thioredoxin is a kind of chaperone, the renaturation that can help IL-11; (3) the reorganization IL-11 of enzyme cutting-out and natural sophisticated IL-11 are only at proline(Pro) of N-terminal deletion; (4) human interleukin 11 of disappearance proline(Pro) proves in vivo and in vitro that with natural IL-11 identical biological function is arranged.Disadvantage with present method is that protease cutting site is enteropeptidase (enterokinase), and the cost height of this enzyme, enzyme are cut efficient not as zymoplasm.This patent has made up novel human interleukin 11 in order to introduce zymoplasm as the proteic proteolytic enzyme of amalgamation and expression point of contact.Zymoplasm differential protein recognition site is Leu-Val-Pro-Arg-Gly-Ser, and the point of contact is between Arg and Gly.In 35 aminoacid sequences of the N-terminal of the IL-11 of analyst and ape three differences (Fig. 1) are only arranged, wherein the difference ape of 6-10 amino acids sequence is: Gly Ser Pro ArgAla, and artificial: Gly Pro Pro Arg Val.
The interleukin 11 of ape just in time is Gly Ser at N-terminal the 6th and 7 amino acids sequences, so just can prepare with having the amalgamation and expression system that contains the zymoplasm restriction enzyme site.Main design is: 5 amino acid of N-terminal that at first lack human interleukin 11 are Pro Gly Pro Pro Pro, the amino acid that then the 7th of the people and the 10th amino acids is replaced to ape promptly is respectively Ser and Ala, and other sequence and people's IL-11 aminoacid sequence is in full accord.This improved Novel Human interleukin-11 can prepare with the amalgamation and expression system that contains the zymoplasm point of contact.The novel recombinant human interleukin 11 of the described method of this patent preparation and design in full accord unnecessary amino acid can not occur.The Novel Human interleukin 11 of using this patent preparation is compared in vivo and in vitro active consistent with the natural human interleukin 11.Because the recombination human interleukin 11 of N-terminal disappearance proline(Pro) formally is applied to the thrombocytopenia that the clinical treatment tumour patient causes because of chemotherapy, the novel improved type interleukin 11 of this patent invention is expected to have clinical value.
The example explanation:
One, the structure of Novel Human interleukin 11 amalgamation and expression system
Select for use the fusion expression plasmid pGEX-4T-1 (Pharmacia) that contains Glutathione S transferase (GST) as the carrier that makes up, this carrier contains the blood coagulation restriction enzyme site.CDNA sequence according to the interleukin 11 of people and ape, and novel improved type human interleukin 11 is 5 amino acid of N-terminal disappearance, the 7th of N-terminal and 10 amino acids change the requirement of Ser and the Ala of ape into, design two pairs of pcr amplification primers, be normal chain: 5 '-CT GGA TCC CCT CGA GCT TCCCCA GAC CCT CGG-3 ', anti-chain 5 '-TTA TCA CAG CCG AGT CTT CAG CAGCAG TAG CCC-3 '.Interleukin-11 whole gene cDNA with the people is a template, the modified version human interleukin 11 gene fragment that amplifies links to each other with the big fragment of pGEX-4T-1 after enzyme is cut, transformed into escherichia coli JM105 filters out to contain and inserts segmental recon called after pGEX-FK-11 (Fig. 2).After sequential analysis, can carry out amalgamation and expression research.
Two, the gene sequencing of Novel Human interleukin 11
From pGEX-FK-11,, link to each other with big fragment behind the Sal I double digestion through BamHI, filter out recon with blue hickie method, called after pUC-FK-11 with pUC-19 with novel improved type human interleukin 11 gene fragment under BamHI and the Sal I double digestion.The gene order (Fig. 3) and the corresponding aminoacid sequence of the Novel Human interleukin-11 that after AB I mdk gene order-checking, obtains inserting, interpretation of result and design in full accord.
Three, the Novel Human interleukin 11 is recombinant expressed
The pGEX-FK-11 transformed into escherichia coli JM105 that contains novel improved type human interleukin 11 gene, in containing the LB substratum of 100ug/ml penbritin, shake bottle and spend the night (37 ℃, 200rpm), contain in the LB substratum of 100ug/ml penbritin 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-FK-11 (44.6kD) that finds to contain the Novel Human interleukin 11 is based on solubility expression, and expression amount accounts for 28% of bacterial protein.
Four, the purifying of recombined new human interleukin 11
Use the 1000ml fermented liquid, about 15 grams of centrifugal collection thalline contain 1%Triton solution suspension thalline with 50mM phosphate buffered saline buffer (PH7.8), at room temperature broken bacterium 20 minutes, and broken bacterium liquid precipitates through ultrasonication centrifugal going after do not have thickness.Supernatant carries out affinitive layer purification through Glutathion-Sepharose (Pharmacia), is adsorbed on the fusion rotein that contains the Novel Human interleukin 11 on the post, cuts with enzyme on the throne under human plasma zymoplasm (every milligram fusion rotein with the 5NIH unit's zymoplasm) room temperature.The recombined new human interleukin 11 that enzyme downcuts, again through the CM-SepharoseCL-4B purifying, the purity of final product surpasses 98%, and the every mg albumen of pyrogen content is lower than 10 EU intracellular toxins.
Five, the physico-chemical property of recombined new human interleukin 11
The recombined new human interleukin 11 molecular weight of present method preparation is 18.6KD, and iso-electric point is greater than 10.0, and the ultraviolet maximum absorption band is 281nm, and the cyanogen bromide peptide figure analysis contains two methionine(Met)s.Amino acid composition analysis is consistent with the Design Theory value, does not wherein have halfcystine.The N-terminal order-checking confirms: Gly Ser Pro Arg Ala Ser Pro Asp Pro Arg Ala Gln Leu Asp SerThr, and in full accord with the N-terminal sequence of the Novel Human interleukin 11 that designs.
Six, the external biological activity of recombined new human interleukin 11
With the cell strain 7TD that depends on interleukin 6
1And T
1165The MTT method survey to live, be the recombination human interleukin 11 of disappearance proline(Pro) of U.S. Genetics Institute and natural human interleukin 11 with reference to product.The specific activity that records the recombined new modified version human interleukin 11 of present method preparation is respectively 2.5 * 10
6(7TD1 cell) and 8.0 * 10
6(T
1165Cell), in full accord with natural specific activity with the human interleukin 11 that lacks proline(Pro).
Seven, platelet increasing number experiment in the recombined new human interleukin 11 body
With the mouse model of 5-FU chemotherapy and the marmoset model of Carboplatin chemotherapy, number of platelets all significantly reduces, the subcutaneous recombined new human interleukin 11 that gives present method preparation, the positive control medicine is the recombination human interleukin 11 of the disappearance proline(Pro) of U.S. Genetics Institute.Results suggest, the recombined new human interleukin 11 has the function of remarkable platelet increasing number, and it increases efficient and positive control (table 1 and Fig. 4) in full accord.
Claims (3)
1, improved human interleukin 11, it is characterized in that it is Pro Gly Pro Pro Pro that its N end lacks 5 amino acid than natural sophisticated human interleukin 11, the amino acid that the 7th of the N-terminal and the 10th amino acids of natural human interleukin-11 replaced to ape interleukin-11 opposite position is respectively Ser and Ala simultaneously.
2, the preparation method of improved human interleukin 11 in the claim 1, it is characterized in that adopting intestinal bacteria amalgamation and expression system, the fusion rotein restriction enzyme site adopts zymoplasm, the fusion rotein purification process adopts affinity chromatography, and the purifying that enzyme is cut the Ro 24-7472/000 of transforming the back adopts CM-Sepharose CL-4B.
3, the application of the improved human interleukin 11 of claim 1 in preparation treatment thrombopenia disease drug.
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| CN 99111463 CN1197874C (en) | 1999-08-16 | 1999-08-16 | Modification and preparation of human interleukin 11 |
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| CN 99111463 CN1197874C (en) | 1999-08-16 | 1999-08-16 | Modification and preparation of human interleukin 11 |
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| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
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| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
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Owner name: CHONGQING MAGEN MEDICINE CO., LTD. Free format text: FORMER OWNER: DUOTAI PHARMACEUTICAL CO., LTD., CHONGQING Effective date: 20060210 |
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| C41 | Transfer of patent application or patent right or utility model | ||
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Effective date of registration: 20060210 Address after: Jiulongpo Branch Park four street 400039 Chongqing 70 block J No. 5 Patentee after: Chongqing MARGAN Pharmaceutical Co. Ltd. Address before: 400041, five floor, biochemical garden, four street, 70 garden, Chongqing hi tech Zone Patentee before: Chongqing Duotai Parmaceutical Co., Ltd. |
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| C19 | Lapse of patent right due to non-payment of the annual fee | ||
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