CN1318644A - Construction process of recombined human single-chain interleukin-12 - Google Patents
Construction process of recombined human single-chain interleukin-12 Download PDFInfo
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- CN1318644A CN1318644A CN 00113786 CN00113786A CN1318644A CN 1318644 A CN1318644 A CN 1318644A CN 00113786 CN00113786 CN 00113786 CN 00113786 A CN00113786 A CN 00113786A CN 1318644 A CN1318644 A CN 1318644A
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Abstract
Interleukin-12 (IL-12) is one known multifunctional cell factor with strongest immune active cell inducing and regulating effect and has activity only when P40 and P35 is in the ratio of 1 to 1 to form allodimer. The present invention provides one simple method to enable to two subunits, P40 and P35, of IL-12 to express commonly and to combine in the 1-to-1 form into recombined human single-chain IL-12 while maintaining original biological activity. The present invention may be embodied in biological engineering field and can be used widely in immunity and tumor treatment.
Description
The present invention relates to a kind ofly prepare the method for interleukin 12 (IL-12), particularly relate to the construction process of a kind of recombined human single-chain interleukin-12 (rhscIL-12) by biotechnology,
Interleukin 12 (IL-12) is known so far immunologically competent cell to be induced the multifunctional cytokine the strongest with regulating effect, natural IL-12 is by the covalently bound heterodimer albumen of disulfide linkage by two polypeptide chains, heavy chain is a P40 albumen, light chain is a P35 albumen, and IL-12 only just had activity at P40 and P35 when ratio formed heterodimer with 1: 1.Some researchists carry out cotransfection by the plasmid that makes up P40 and P35 subunit respectively or make up the carrier that contains P40 and P35 Expression element respectively in single plasmids before the present invention, what have also that human contains internal ribosome entry site sequence transcribes that the polycistronic transcription sublist reaches P40 and P35 in the virus, or E1 district and E3 district that P40 and P35cDNA insert recombinant adenoviral vector are respectively expressed.These methods are not only complicated, and are difficult to reach P40 and P35 forms heterodimer, the activated heterodimer albumen of uncontrollable generation with 1: 1 ratio.
The purpose of this invention is to provide a kind of P40 that simply makes IL-12 and two subunits of P35 can co expression, and can make P40 and P35 carry out the construction process of the recombined human single-chain interleukin-12 (rhscIL-12) of nature combination with 1: 1 form.
The objective of the invention is to realize in the following manner: a kind of construction process of recombined human single-chain interleukin-12, the clone who comprises hIL-12 (human interleukin 12) P40 and the cDNA of P35 subunit, the gene fusion of hIL-12 P40 and P35 subunit, rhscIL-12 (recombined human single-chain interleukin-12) Construction of eukaryotic, it is characterized in that: by the recombinant PCR technology with 5 of 3 of the P40 subunit coding region cDNA sequence of hIL-12 ' end and P35 subunit coding region cDNA sequence ' hold, manual splice (Linker) by a hydrophobicity polypeptide is spliced, make up rhIL-12 (recombinant human interleukin 12) P40-Linker-P35 strand fusion gene, and expression obtains rhscIL-12 (recombined human single-chain interleukin-12) in eukaryotic cell.
Concrete grammar is: stimulate people B lymphoblast strain NC37 cell extraction mRNA with PDBu, with RT-PCR technology amplification hIL-12P40 and the cDNA of P35 subunit, with 5 of 3 ' end of the P40 subunit coding region cDNA sequence that obtains and P35 subunit coding region cDNA sequence ' hold, manual splice---fifteen amino acid residue with a coding hydrophobicity polypeptide, splice by the recombinant PCR technology, construct rhIL-12P40-Linker-P35 strand fusion gene, insert the pcDNA3.1 eukaryon expression plasmid, make up rhscIL-12 (recombined human single-chain interleukin-12) fusion gene carrier for expression of eukaryon, and change mammalian cell cos7 over to, express activated rhseIL-12.
Compared with the prior art, outstanding advantage of the present invention is to have adopted the i.e. peptide linker of 15 amino-acid residues of generally acknowledged Linker, (it has good kindliness and folding property, and have enough length and guarantee that strand IL-12 fusion rotein forms and the similar space conformation of natural IL-12,) by recombinant PCR 3 ends of P40 and 5 ends of P35 are spliced, construct rhIL-12P40-Linker-P35 strand fusion gene, make P40 and P35 form heterodimer with 1: 1 matched form, guaranteed the activity of IL-12, and can be under the control of a promotor accurate translation activated protein, reach the purpose that control produces heterodimer.The present invention can implement at the production unit of bioengineering field, and product-recombined human single-chain interleukin-12 (rhscIL-12) that enforcement the present invention obtains can be widely used in immunity and struma treatment of diseases.
Animal body experiment showed, that outward rhscIL-12 has the T of enhancing lymph small cell proliferative activity, stimulates the T lymphocyte to produce Interferon, rabbit, stimulating natural killer hyperplasia, strengthens natural killer cell activity, suppresses simple herpesvirus replication, suppresses the outgrowth function of hepatoma cell line.Animal experiment showed, that at body rhscIL-12 can make the transplantability people liver cancer of nude mice disappear fully.In the experimentation, do not find that the rhscIL-12 that uses has any toxicity to nude mice.
Embodiment:
One, the clone of hIL-12 (Ro 24-7472/000-12) P40 and the cDNA of P35 subunit: Nc37 cell strain cultivation → Nc37 cell mRNA is extracted---synthetic IL-12P40 of RT-PCR and P35cDNA (concrete steps are undertaken by the Access RT-PCR system of Promege company explanation).Insert the pGEM-Teasy carrier respectively.
Two, recombined human single-chain IL-12 (rhscIL-12) fusion gene makes up:
RhscIL-12P40 constitutes people's strand IL-12 fusion gene (rhscIL-12) with the cDNA of P35 subunit at the hydrophobicity polypeptid DNA of a coding tool flexibility under segmental the connection.The inner primer that is used for recombinant PCR is according to the Linker sequence, and P40 3 terminal sequences, P35 5 sequences design, and carry out gene recombination with overlapping extension principle.Linker is the hydrophobicity polypeptide of fifteen amino acid residue.Concrete steps: with PCR increase respectively hIL-12P35 and P40cDNA, with pGMET-P40 and pGEMT-P35 plasmid is template, increase with outer primer in P40 and the P35 respectively, and in eukaryotic cell, express, obtain to be added with P40 and P35PCR product---the recombined human single-chain IL-12 (rhscIL-12) of Linker complementary sequence.
Claims (1)
1, a kind of construction process of recombined human single-chain interleukin-12, the clone who comprises hIL-12 (human interleukin 12) P40 and the cDNA of P35 subunit, the gene fusion of hIL-12 P40 and P35 subunit, rhscIL-12 (recombined human single-chain interleukin-12) Construction of eukaryotic, it is characterized in that: by the recombinant PCR technology with 5 of 3 of the P40 subunit coding region cDNA sequence of hIL-12 ' end and P35 subunit coding region cDNA sequence ' hold, manual splice (Linker) by a hydrophobicity polypeptide is spliced, make up rhIL-12 (recombinant human interleukin 12) P40-Linker-P35 strand fusion gene, and expression obtains rhscIL-12 (recombined human single-chain interleukin-12) in eukaryotic cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00113786 CN1318644A (en) | 2000-04-18 | 2000-04-18 | Construction process of recombined human single-chain interleukin-12 |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 00113786 CN1318644A (en) | 2000-04-18 | 2000-04-18 | Construction process of recombined human single-chain interleukin-12 |
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| CN1318644A true CN1318644A (en) | 2001-10-24 |
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| CN 00113786 Pending CN1318644A (en) | 2000-04-18 | 2000-04-18 | Construction process of recombined human single-chain interleukin-12 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002495A (en) * | 2009-08-31 | 2011-04-06 | 成都蓉生药业有限责任公司 | Expression cassette and expression carrier for expressing heterodimer glycoprotein hormones and preparation method thereof |
| CN105218682A (en) * | 2015-10-26 | 2016-01-06 | 深圳精准医疗科技有限公司 | Through tumor therapeutic agent and method for making and the purposes of the transformation of IL-12/CD62L fusion rotein |
| CN105585637A (en) * | 2015-12-23 | 2016-05-18 | 深圳精准医疗科技有限公司 | Tumor therapeutic agent based on IL-12 stable membrane expression as well as preparation method and application of tumor therapeutic agent |
| CN106381309A (en) * | 2016-08-31 | 2017-02-08 | 广东科玮生物技术股份有限公司 | Expression vector of human recombinant interleukin-12 |
| EP3083666A4 (en) * | 2013-12-18 | 2017-09-20 | Intrexon Corporation | Single chain il-12 nucleic acids, polypeptids, and uses thereof |
| CN108064176A (en) * | 2015-04-22 | 2018-05-22 | 库瑞瓦格股份公司 | For treating the composition containing RNA of tumor disease |
| CN108424916A (en) * | 2018-03-29 | 2018-08-21 | 中国水产科学研究院南海水产研究所 | Flower perch interleukins IL-12p40 genes and application thereof |
| CN109517052A (en) * | 2018-12-04 | 2019-03-26 | 江苏禄亿生生物科技有限公司 | A kind of IL-12 protein mutant and its preparation method and application, NK cell culture system and the method for cultivating NK cell |
-
2000
- 2000-04-18 CN CN 00113786 patent/CN1318644A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002495B (en) * | 2009-08-31 | 2012-07-18 | 成都蓉生药业有限责任公司 | Expression cassette and expression carrier for expressing heterodimer glycoprotein hormones and preparation method thereof |
| CN102002495A (en) * | 2009-08-31 | 2011-04-06 | 成都蓉生药业有限责任公司 | Expression cassette and expression carrier for expressing heterodimer glycoprotein hormones and preparation method thereof |
| EP3083666A4 (en) * | 2013-12-18 | 2017-09-20 | Intrexon Corporation | Single chain il-12 nucleic acids, polypeptids, and uses thereof |
| US10918740B2 (en) | 2015-04-22 | 2021-02-16 | Curevac Ag | RNA containing composition for treatment of tumor diseases |
| US10869935B2 (en) | 2015-04-22 | 2020-12-22 | Curevac Ag | RNA containing composition for treatment of tumor diseases |
| CN108064176A (en) * | 2015-04-22 | 2018-05-22 | 库瑞瓦格股份公司 | For treating the composition containing RNA of tumor disease |
| CN105218682B (en) * | 2015-10-26 | 2019-05-07 | 杨晶 | The tumor therapeutic agent and its preparation method and purposes being transformed through IL-12/CD62L fusion protein |
| CN105218682A (en) * | 2015-10-26 | 2016-01-06 | 深圳精准医疗科技有限公司 | Through tumor therapeutic agent and method for making and the purposes of the transformation of IL-12/CD62L fusion rotein |
| WO2017071173A1 (en) * | 2015-10-26 | 2017-05-04 | 杨世成 | Tumor therapeutic agent modified by il-12/cd62l fusion protein and preparation method and use thereof |
| CN105585637B (en) * | 2015-12-23 | 2020-04-03 | 杨晶 | Tumor therapeutic agent based on IL-12 stable membrane expression and preparation method and application thereof |
| WO2017107353A1 (en) * | 2015-12-23 | 2017-06-29 | 杨世成 | Cancer treatment agent, preparation method and use thereof employing il-12 with stable membrane expression |
| CN105585637A (en) * | 2015-12-23 | 2016-05-18 | 深圳精准医疗科技有限公司 | Tumor therapeutic agent based on IL-12 stable membrane expression as well as preparation method and application of tumor therapeutic agent |
| CN106381309A (en) * | 2016-08-31 | 2017-02-08 | 广东科玮生物技术股份有限公司 | Expression vector of human recombinant interleukin-12 |
| CN108424916A (en) * | 2018-03-29 | 2018-08-21 | 中国水产科学研究院南海水产研究所 | Flower perch interleukins IL-12p40 genes and application thereof |
| CN108424916B (en) * | 2018-03-29 | 2021-04-30 | 中国水产科学研究院南海水产研究所 | Lateolabrax japonicus interleukin IL-12p40 gene and application thereof |
| CN109517052A (en) * | 2018-12-04 | 2019-03-26 | 江苏禄亿生生物科技有限公司 | A kind of IL-12 protein mutant and its preparation method and application, NK cell culture system and the method for cultivating NK cell |
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