Method for preparing medicine using silkworm expressed human beta interferon

A technology for preparation of interferon-beta and drugs, which is applied in the field of recombinant production of human interferon-beta, can solve the problems of no oral dosage form and high production cost, achieve high clinical application value, relieve pain and the threat of infection

Inactive Publication Date: 2003-03-26
特菲(天津)生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, IFNβ is mainly expressed in Escherichia coli (Remant et al., 1983; Goeddel et al., PNAS, 1980, VOL77, 4003...

Method used

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  • Method for preparing medicine using silkworm expressed human beta interferon
  • Method for preparing medicine using silkworm expressed human beta interferon
  • Method for preparing medicine using silkworm expressed human beta interferon

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Experimental program
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Effect test

Embodiment 1

[0014] Take Chinese fibroblasts and grind them at low temperature, add 1ml of Trizol RNA extraction solution produced by GIBCOBRL company, shake gently for 10 minutes, add 500μl of chloroform (Zhejiang Dier Pharmaceutical Co., Ltd.), place at room temperature for 10 minutes, and centrifuge at 12000rpm for 10 minutes. Minutes, take the supernatant, add 2 times the volume of ethanol, after mixing, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 1000 units of reverse transcriptase (GIBCOBRL company) and 4dNTP (GIBCOBRL company) for reverse transcription, 37 ℃ , 1 hour to obtain cDNA synthesized by reverse transcription of mRNA. Primers were designed according to the published human beta interferon gene sequence (Proc.Natl.Acad.Sci.USA, 1980, VOL77, 4003), and BamHI and PstI sites were designed at the 5' and 3' ends, respectively. The upstream primer is: 5'GGGGATCCATGAGCTACAACTTACTTGGA, the downstream primer is: 5'CCCTGCAGTCAGTTTCGGACGTAACCTGTAAG, and the human...

Embodiment 2

[0015] Embodiment 2, the silkworm baculovirus transfer plasmid of human beta interferon gene

[0016]The plasmid pUC-β containing the human interferon beta gene fragment was digested by BamHI and PstI, and then connected to the transfer vector pBacPAK8 (CLONTECH Company) which was digested by BamHI and PstI to obtain the recombinant transfer vector plasmid pBacIFN-β ( figure 2 ). The gene was identified to be correct by restriction analysis. Embodiment 3, the acquisition of the recombinant baculovirus of human beta interferon gene

Embodiment 3

[0017] Take 5 ul of the insect baculovirus transfer plasmid pBacIFN-β containing the human β-interferon gene and 6 ul of the modified virus BmBacPAK6 linearized by AocI digestion (constructed by the applicant), add 100 ul of serum-free TC-100 medium and mix well. Take 6ul Dosper (Bowringman Company) and add 100ul serum-free TC-100 medium (GIBCOBRL Company) and mix well. Wash the BmN cells previously cultured in 35mm plates (preserved strain of the Institute of Biochemistry, Zhejiang University) twice with serum-free TC-100 medium, add the pBacIFN-β transfer plasmid and Dosper mixture dropwise, and incubate at 27°C After 4-5 days, the supernatant was collected for the first round of plaque screening. Take 5ul supernatant to infect Bm N cells in a 35mm plate, discard the supernatant after 1 hour and add equal volumes 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 fo...

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Abstract

The invention refers to a method of using the bacilliform reformed virus with human beta-interferon gene to infect the silkworm grub and chrysalis in order to produce the human beta interferon and the prepared oral drugs. It includes the following: utilize the silkworm bacilliform virus-expressing system to reform the human beta-interferon gene for obtaining the reformed virus; inoculate the silkworm grub and chrysalis and the reformed virus for expression, and after separation, purification, refrigeration and desiccation, make them into oral drugs which has the remarkable action on the second-type hepatitis virus by the axamination on animals.

Description

technical field [0001] The present invention relates to the method for recombination producing human beta interferon, more specifically, the present invention relates to the method that the recombinant baculovirus containing human beta interferon gene infects silkworm larvae and pupae to produce human beta interferon, and the present invention also relates to expressing The silkworm preparation method of human beta interferon. Background technique [0002] Interferon is a cytokine with dual effects of anti-tumor and anti-virus. Interferon β (interferonβ, IFNβ, IFN-β) is encoded by a single gene, with a signal peptide of 21 amino acids and a mature protein of 166 amino acids. The coding gene is located on chromosome 9 (Derynck et al., Nature, 1980, 285; Taniguch et al., 1980 ) Unlike human IFNα, IFNβ has a potential N-glycosylation site at position 80, and the mature protein is also glycosylated. IFNβ shares the same receptor with IFNα, and its biological activity is simila...

Claims

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Application Information

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IPC IPC(8): A61K38/21C07K14/565C12N7/01C12N15/09C12N15/10C12N15/22C12N15/70C12N15/866C12N15/88
Inventor 张耀洲金勇丰吴祥甫
Owner 特菲(天津)生物医药科技有限公司
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