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Method for using silkworm to express human interleukin 11 to produce medicine

An interleukin and drug technology, which is applied in biochemical equipment and methods, botanical equipment and methods, and drug combinations, etc., can solve the problem of high production costs, and achieve the effects of perfect processing, high expression efficiency and stable gene expression products.

Inactive Publication Date: 2003-04-09
浙江中奇生物药业股份有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

Since the successful molecular cloning of IL-11 in 1990, the gene structure and biological functions of IL-11 have been elucidated successively, and have been expressed in mammalian cells and Escherichia coli successively (Kitts et al., 1993, Liu Beiling et al., 1996) , but it is basically an injection drug, the production cost is high, and there is basically no oral dosage form on the market

Method used

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  • Method for using silkworm to express human interleukin 11 to produce medicine
  • Method for using silkworm to express human interleukin 11 to produce medicine
  • Method for using silkworm to express human interleukin 11 to produce medicine

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Embodiment 1

[0015]Primers were designed according to the published human interleukin-11 gene sequence (PNAS, 1990, VOL87, 7512), and XhoI and BamHI sites were designed at the 5 and 3 ends, respectively. The upstream primer is: 5'GGGCTCGAGATGCCTGGGCCACCACCTGGC3', and the downstream primer is: 5'GGGGATCCTCACAGCCGAGTCTTCAG3'. Take human bone marrow tissue cells, grind them at low temperature, add 1ml of Trizol RNA extraction solution produced by GIBCOBRL Company, shake gently for 10 minutes, and then add 500 μl of chloroform (Zhejiang Dier Pharmaceutical Co., Ltd.), placed at room temperature for 10 minutes, centrifuged at 12,000 rpm for 10 minutes, took the supernatant, added 2 times the volume of ethanol, mixed well, centrifuged at 12,000 rpm for 10 minutes, discarded the supernatant, and added reverse transcriptase 1000 Unit (GIBCOBRL) and 4dNTP (GIBCOBRL) were reverse transcribed at 37°C for 1 hour to obtain cDNA synthesized by reverse transcription of mRNA. Human interleukin-11 gene was...

Embodiment 2

[0016] The recombinant plasmid pUCIL-11 was double-digested with restriction endonucleases XhoI and BamHI to obtain the hIL-11 cDNA fragment added with the XhoI restriction site, which was inserted into the XhoI and XhoI of the transfer vector plasmid pBacPAK8 (CLONTECH Company) Between the two sites of Bgl II, the bacIL-11 transfer plasmid pBacIL-11 containing hIL-11 gene was constructed ( figure 1 ). Embodiment 3, the acquisition of the recombinant baculovirus of hIL-11 gene

Embodiment 3

[0017] Take 5ul insect baculovirus transfer plasmid pBacIL-11 containing human interleukin-11 gene and 6ul wild silkworm nuclear polyhedrosis virus DNA for co-transfection. Take 6ul Lipofectin (GIBCOBRL company) and add 100ul serum-free TC-100 medium and mix well. The BmN cells previously cultured in a 35mm Dish were washed twice with serum-free TC-100 (GIBCOBRL company) medium, and the transfer plasmid and Lipofectin mixture was added dropwise, cultured at 27°C for 4-5 days, and the supernatant was collected for the second stage. One round of plaque screening. Take 5ul of the supernatant to infect the BmN cells in a 35mm Dish, discard the supernatant after 1 hour and add an equal amount of mixed TC-100 medium and low melting point agarose. Pick plaques after 4-5 days, infect BmN cells for 3-4 days, save the supernatant, lyse the cells with NaOH for Southern hybridization, and take the supernatant of positive clones for 8 rounds of plaque screening and Southern hybridization ...

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Abstract

The present invention belongs to the field of gene engineering technology of producing polypeptide medicine in biological pharmaceutical engineering. Via Bombyx mori baculvirus expression system, human interleukin 11 gene is rcombined to obtain recombinant virus, which is preserved in China Microbial Strain Preservation Committee in the preservation number of CMCC No.0602 through inoculating the recombinant virus to Bombyx mori cell, larva and pupa to express, separation and purification and freezing drying, injection and oral liquid are produced. Animal test shows that the present invention has obvious blood platelet increasing function.

Description

technical field [0001] The present invention relates to a method for preparing human interleukin (IL-11) from silkworm larvae and pupae through the Bombyx mori baculovirus expression system. The present invention also relates to the production of human interleukin (IL-11) by silkworm larvae and pupae Methods of thrombotic drugs. Background technique [0002] Interleukin (IL-11) is a multifunctional cytokine derived from myelofibroblasts in the mammalian hematopoietic microenvironment (Du et al., 1994). It can stimulate the proliferation of IL-6-dependent cell lines, and has a stimulating effect on the secretion of B cell antibodies that depend on T cells (Paul et al., 1992); it cooperates with IL-3 to promote the formation and maturation of human and mouse megakaryocytes, and promotes Proliferation of primitive progenitor cells, increase platelet count, promote erythropoiesis, induce acute phase protein secretion in the liver, act on the hematopoietic microenvironment, inhi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/64A61K38/20A61P7/02C12N15/26C12N15/85
Inventor 张耀洲金勇丰吴祥甫郭锡杰
Owner 浙江中奇生物药业股份有限公司
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