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Method for producing medicine using silkworm expressed numan epidermal growth factor

A technology of epidermal growth factor and expression products, which is applied in the field of expressing human epidermal growth factor to produce drugs, and can solve the problem of high production cost

Inactive Publication Date: 2004-10-13
正源堂(天津)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, EGF is mainly expressed in Escherichia coli (Yadwad et al., 1989; Shimizum et al., 1991). It is basically an injection drug, and the production cost is high. There is basically no oral dosage form on the market.

Method used

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  • Method for producing medicine using silkworm expressed numan epidermal growth factor
  • Method for producing medicine using silkworm expressed numan epidermal growth factor
  • Method for producing medicine using silkworm expressed numan epidermal growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1, artificial chemical synthesis of human EGF gene and construction of Escherichia coli vector pSK-EGF

[0016] According to the reported EGF gene sequence (Proc.Natl.Acad.Sci.USA, 1983, VOL80,7462), synthesize 4 oligonucleotide chains of encoding EGF with DNA synthesizer and phosphite triester method, use poly Separation and purification by acrylamide-7mol / L urea denaturing gel electrophoresis, the sequences are: oligonucleotide chain 1: 5'-GGA ATT CGT TAA CTC CGA CTC CGA ATG TCC ATT GTCCCA CGA CGG TTA CTG TTT GCA CGA-3 ', oligonucleotide strand 2: 5'-AAGCTT TGG ACA AGT ACG CCT GTA ACT GTG TTG TTG GTT ACATCG GTG AAA GAT GTC AAT A-3, oligonucleotide strand 3: 5'-GGA ATT CTCATT ATT CCC ACC ACT TCA AGT CTC TGT ATT GA ATC TTT CACCGA TGT-3, oligonucleotide strand 4: 5'-GGC GTA CTT GTC CAA AGC TTCGAT GTA CAT ACA AAC ACC GTC GTG CAA ACA GTA ACCGTC-3', oligonucleotide Phosphorylation of the 5' end of the polynucleotide chain, renaturation, chain extension, ligation...

Embodiment 2

[0017] Embodiment 2, the construction of the Bombyx mori baculovirus transfer plasmid of human EGF gene

[0018] After the plasmid pSK-EGF containing the human EGF gene fragment was digested by BamHI (Boringman Company) and EcoRV, the gene fragment was recovered with low-melting point glue, and transferred with the transfer vector pBacPAK8 (CLONTECH Company) double-digested by BamHI and SmaI. connection to obtain the recombinant transfer vector plasmid pBacEGF ( figure 1 ), the gene was identified to be correct by restriction analysis.

Embodiment 3

[0019] Embodiment 3, the acquisition of the recombinant baculovirus of human EGF gene

[0020]Get 5 ul of the silkworm baculovirus transfer plasmid pBacEGF containing human EGF gene and 6 ul of the modified virus BmBacPAK6 (constructed by the applicant) through AocI (Boringman Company) enzyme digestion and linearization, and add 100 ul of serum-free TC-100 (GIBCOBRL Company ) culture medium was mixed evenly. Take 6ul Dosper (Bowringman Company) and add 100ul serum-free TC-100 medium and mix well. Wash the BmN cells previously cultured in a 35mm dish (preserved strain of the Institute of Biochemistry, Zhejiang University) twice with serum-free TC-100 medium, add the transfer plasmid and Dosper mixture dropwise, and culture at 27°C for 4-5 days , the supernatant was collected for the first round of plaque screening. Take 5 ul supernatant to infect silkworm cells in a 35mm plate, discard the supernatant after 1 hour and add an equal amount of mixed TC-100 medium and low melting...

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Abstract

The invention belongs to the technique field of producing multi-peptide drugs by gene engineering. It clones human EGF gene into the silkworm bacilliform virus (Bombyx mori Nuclear polyhedrosis Virus) transferring carrier pBacPAK8 and makes them carry through homologous reformation in the silkworm cells to obtain the reformed virus BmBacEGF with human-coat growing gene. Use the reformed virus to to inocualte the silkworm grub and chrysalis by acupuncture in order to make the human-coat growing gene efficient expressed. Then make the grub and chrysalis into oral drugs which has remarkable action on the digestible ulcer by examination on animal.

Description

technical field [0001] The invention relates to a method for producing medicine by using silkworms to express human epidermal growth factor (Epidermal growth Factor, EGF). Background technique [0002] Epidermal growth factor (EGF) was accidentally discovered by Cohen in 1959 when studying nerve growth factor (NGF), and was isolated from mouse submandibular gland in 1962. In vivo and in vitro experiments have proved that it has the function of promoting proliferation of epithelial cells derived from various tissues. Human epidermal growth factor was first purified from human urine by Gregory et al. in 1975. Through research, it was found that it has the same biological activity as mouse EGF . The most potential use of EGF is in clinical application. EGF has the function of promoting epithelial regeneration, and can be used to promote the healing of surgical wounds and other wounds, such as skin grafts, skin abrasions, burns, erosion, etc.; to promote corneal epithelial rege...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/18A61P1/04C07K14/485C12N7/01C12N15/09C12N15/10C12N15/12C12N15/866C12N15/88
Inventor 张耀洲金勇丰吴祥甫
Owner 正源堂(天津)生物科技有限公司
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