CN1318644A - Construction process of recombined human single-chain interleukin-12 - Google Patents
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- 102000013462 Interleukin-12 Human genes 0.000 title claims abstract description 28
- 108010065805 Interleukin-12 Proteins 0.000 title claims abstract description 28
- 229940117681 interleukin-12 Drugs 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 7
- 238000010276 construction Methods 0.000 title claims description 7
- 239000002299 complementary DNA Substances 0.000 claims description 14
- 229940040731 human interleukin-12 Drugs 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 230000004927 fusion Effects 0.000 claims description 9
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- 229920001184 polypeptide Polymers 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
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- 108010050904 Interferons Proteins 0.000 description 1
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Abstract
Description
本发明涉及一种由生物技术制备白细胞介素12(IL-12)的方法，特别是涉及一种重组人单链白细胞介素-12(rhscIL-12)的构建方法，The present invention relates to a method for preparing interleukin 12 (IL-12) by biotechnology, in particular to a method for constructing recombinant human single-chain interleukin-12 (rhscIL-12),
白细胞介素12(IL-12)是至今已知对免疫活性细胞诱导和调节作用最强的多功能细胞因子，天然IL-12是由两条多肽链通过二硫键共价结合的异二聚体蛋白，重链为P40蛋白，轻链为P35蛋白，IL-12只有在P40和P35以1∶1比例形成异二聚体时才有活性。本发明前一些研究人员通过分别构建P40和P35亚基的质粒进行共转染或在单一质粒中构建分别含P40和P35表达元件的载体，也有人用含有内部核糖体进入位点序列的转录病毒中多顺反子转录子表达P40和P35，或将P40和P35cDNA分别插入重组腺病毒载体的E1区和E3区进行表达。这些方法不但复杂，而且很难达到P40和P35以1∶1比例形成异二聚体，无法控制产生有活性的异二聚体蛋白。Interleukin 12 (IL-12) is the multifunctional cytokine known so far to have the strongest induction and regulation effect on immune active cells. Natural IL-12 is a heterodimerization of two polypeptide chains covalently bonded by disulfide bonds. IL-12 is active only when P40 and P35 form a heterodimer at a ratio of 1:1. Before the present invention, some researchers carried out co-transfection by respectively constructing plasmids of P40 and P35 subunits or constructing vectors containing P40 and P35 expression elements in a single plasmid, and some people used transcription viruses containing internal ribosome entry site sequences The polycistronic transcript expresses P40 and P35, or inserts the P40 and P35 cDNA into the E1 region and E3 region of the recombinant adenovirus vector for expression. These methods are not only complicated, but also difficult to achieve P40 and P35 to form heterodimers at a ratio of 1:1, and cannot control the production of active heterodimeric proteins.
本发明的目的是提供一种简单的使IL-12的P40和P35两条亚基能共同表达、并能使P40和P35以1∶1形式进行自然组合的重组人单链白细胞介素-12(rhscIL-12)的构建方法。The purpose of the present invention is to provide a simple recombinant human single-chain interleukin-12 that can co-express the two subunits of IL-12, P40 and P35, and can naturally combine P40 and P35 in a 1:1 form (rhscIL-12) construction method.
本发明的目的是通过以下方式实现的：一种重组人单链白细胞介素-12的构建方法，包括hIL-12(人白细胞介素12)P40和P35亚单位cDNA的克隆、hIL-12 P40和P35亚单位的基因融合、rhscIL-12(重组人单链白细胞介素-12)真核表达载体的构建，其特征在于：通过重组PCR技术将hIL-12的P40亚单位编码区cDNA序列的3′端和P35亚单位编码区cDNA序列的5′端，通过一个疏水性多肽的人工接头(Linker)进行拼接，构建rhIL-12(重组人白细胞介素12)P40-Linker-P35单链融合基因，并在真核细胞中表达得到rhscIL-12(重组人单链白细胞介素-12)。The object of the present invention is achieved in the following manner: a construction method of recombinant human single-chain interleukin-12, including the cloning of hIL-12 (human interleukin 12) P40 and P35 subunit cDNA, hIL-12 P40 Gene fusion with P35 subunit, construction of rhscIL-12 (recombinant human single-chain interleukin-12) eukaryotic expression vector, characterized in that: the cDNA sequence of the P40 subunit coding region of hIL-12 is obtained by recombinant PCR technology The 3' end and the 5' end of the cDNA sequence of the P35 subunit coding region are spliced through a hydrophobic polypeptide artificial linker (Linker) to construct rhIL-12 (recombinant human interleukin 12) P40-Linker-P35 single-chain fusion Gene, and expressed in eukaryotic cells to obtain rhscIL-12 (recombinant human single-chain interleukin-12).
具体方法是：以PDBu刺激人B淋巴母细胞株NC37细胞提取mRNA，以RT-PCR技术扩增hIL-12P40和P35亚单位cDNA，将获得的P40亚单位编码区cDNA序列的3′端和P35亚单位编码区cDNA序列的5′端，用一个编码疏水性多肽的人工接头——15氨基酸残基，通过重组PCR技术进行拼接，构建出rhIL-12P40-Linker-P35单链融合基因，插入pcDNA3.1真核表达质粒，构建rhscIL-12(重组人单链白细胞介素-12)融合基因真核表达载体，并转入哺乳动物细胞cos7，表达有活性的rhseIL-12。The specific method is: stimulate human B lymphoblastoid cell line NC37 cells with PDBu to extract mRNA, amplify hIL-12P40 and P35 subunit cDNA by RT-PCR technology, and combine the 3′ end of the obtained P40 subunit coding region cDNA sequence with the P35 The 5′ end of the cDNA sequence of the subunit coding region was spliced with an artificial linker encoding a hydrophobic polypeptide—15 amino acid residues by recombinant PCR technology to construct a rhIL-12P40-Linker-P35 single-chain fusion gene, which was inserted into pcDNA3 .1 Eukaryotic expression plasmid, construct rhscIL-12 (recombinant human single-chain interleukin-12) fusion gene eukaryotic expression vector, and transfer it into mammalian cell cos7 to express active rhseIL-12.
与已有技术相比，本发明的突出优点是采用了公认的Linker即15个氨基酸残基的多肽接头，(它具有良好的柔顺性和折叠性，并具有足够的长度保证单链IL-12融合蛋白形成与天然IL-12类似的空间构象，)通过重组PCR对P40的3端和P35的5端进行拼接，构建出rhIL-12P40-Linker-P35单链融合基因，使P40和P35以1∶1的匹配形式形成异二聚体，保证了IL-12的活性，并可在一个启动子的控制之下翻译表达活性蛋白，达到控制产生异二聚体的目的。本发明可在生物工程领域的生产单位实施，实施本发明得到的产品—重组人单链白细胞介素-12(rhscIL-12)可广泛应用于免疫和瘤肿疾病的治疗。Compared with the prior art, the outstanding advantage of the present invention is that it adopts the recognized Linker, which is a polypeptide linker of 15 amino acid residues, (it has good flexibility and foldability, and has sufficient length to ensure single-chain IL-12 The fusion protein forms a spatial conformation similar to natural IL-12.) The 3-end of P40 and the 5-end of P35 were spliced by recombinant PCR to construct a rhIL-12P40-Linker-P35 single-chain fusion gene. The matching form of :1 forms a heterodimer, which ensures the activity of IL-12, and can translate and express the active protein under the control of a promoter, so as to achieve the purpose of controlling the production of heterodimer. The present invention can be implemented in production units in the field of bioengineering, and the product obtained by implementing the present invention—recombinant human single-chain interleukin-12 (rhscIL-12) can be widely used in the treatment of immunity and tumor diseases.
动物体外实验证明，rhscIL-12具有增强T淋巴细细胞增生活性、刺激T淋巴细胞产生干扰素、刺激自然杀伤细胞增生、增强自然杀伤细胞活性、抑制单纯疱疹病毒复制、抑制肝癌细胞系增生的功能。动物在体实验证明，rhscIL-12可使裸鼠的移植性人肝癌完全消退。实验过程中，未发现所使用rhscIL-12对裸鼠有任何毒性。Animal experiments in vitro have proved that rhscIL-12 has the functions of enhancing the proliferation of T lymphocytes, stimulating T lymphocytes to produce interferon, stimulating the proliferation of natural killer cells, enhancing the activity of natural killer cells, inhibiting the replication of herpes simplex virus, and inhibiting the proliferation of liver cancer cell lines . Animal experiments in vivo proved that rhscIL-12 can completely regress transplanted human liver cancer in nude mice. During the experiment, the rhscIL-12 used was not found to have any toxicity to nude mice.
实施例：Example:
一、hIL-12(人白细胞介素-12)P40和P35亚单位cDNA的克隆：Nc37细胞株培养→Nc37细胞mRNA提取——RT-PCR合成IL-12P40和P35cDNA(具体步骤按Promege公司Access RT-PCR system说明进行)。分别插入pGEM-Teasy载体。1. Cloning of hIL-12 (human interleukin-12) P40 and P35 subunit cDNA: Nc37 cell line culture→Nc37 cell mRNA extraction——RT-PCR synthesis of IL-12P40 and P35cDNA (specific steps according to Promege Company Access RT -PCR system instructions). Inserted into pGEM-Teasy vector respectively.
二、重组人单链IL-12(rhscIL-12)融合基因构建：2. Construction of recombinant human single-chain IL-12 (rhscIL-12) fusion gene:
rhscIL-12P40和P35亚单位cDNA在一编码具柔性的疏水性多肽DNA片段的连接下构成人单链IL-12融合基因(rhscIL-12)。用于重组PCR的内引物根据Linker序列，P40 3端序列、P35 5序列进行设计，以overlapping extension原理进行基因重组。Linker为15氨基酸残基的疏水性多肽。具体步骤：以PCR分别扩增hIL-12P35和P40cDNA，以pGMET-P40和pGEMT-P35质粒为模板，分别用P40和P35内外引物进行扩增，并在真核细胞中表达，获得加有Linker互补序列的P40和P35PCR产物——重组人单链IL-12(rhscIL-12)。The cDNA of rhscIL-12P40 and P35 subunits is connected by a DNA fragment encoding a flexible hydrophobic polypeptide to form a human single-chain IL-12 fusion gene (rhscIL-12). The internal primers used for recombination PCR were designed according to the Linker sequence, P40 3-terminal sequence, and P35 5-terminal sequence, and gene recombination was carried out based on the principle of overlapping extension. Linker is a hydrophobic polypeptide with 15 amino acid residues. Specific steps: amplify hIL-12P35 and P40 cDNA by PCR respectively, use pGMET-P40 and pGEMT-P35 plasmids as templates, respectively amplify with internal and external primers of P40 and P35, and express in eukaryotic cells to obtain complementary DNA with Linker P40 and P35 PCR products of the sequence - recombinant human single-chain IL-12 (rhscIL-12).
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 | Compositions containing RNA for the treatment of tumor diseases |
| 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 |
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2000
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 | Compositions containing RNA for the treatment of tumor diseases |
| 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 stable membrane expression of IL-12 and its preparation method and use |
| 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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