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Method for preparing recombinant vEGF using domestic silkworm as biological factory

A silkworm and biological technology, applied in protein purification and activity analysis, and DNA recombination technology, can solve the problems of unstable yeast plasmid transformants and high cost of mammalian cell culture, and achieve the effect of rapid extraction and high biological activity

Inactive Publication Date: 2006-05-17
ZHEJIANG UNIV
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  • Summary
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  • Application Information

AI Technical Summary

Problems solved by technology

However, yeast plasmid transformants are unstable and expensive to produce in cultured mammalian cells

Method used

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Embodiment Construction

[0016] 1. Materials: RNA extraction kit was purchased from Qiagene. DNA processing and PCR amplification kits were purchased from Takara Biomedicals (Kyoto, Japan). TA cloning kit, baculovirus transfer vector pBlueBacHis, lipofectin and Ni-NTA resin are all products of Invitrogen. DNA sequencing kits were purchased from PE Applied Biosystems. The hybrid virus HyNPV between BmNPV and AcNPV is newly constructed by ourselves. Fetal calf serum FCS, medium TC-100 for Spodoptera frugiperda cell line Sf21 and medium 199 containing Earle’s salt for culturing human umbilical vein endothelial cells HUVEC are all products of GibcoBRL. Medium ESF921 for the Fall Armyworm cell line Tn-5 was purchased from Expressionsystems. Human umbilical vein endothelial cells HUVEC, SCS and ECGS are all products of Technoclone GmbH. Spodoptera frugiperda cells Sf21 were cultured at 27°C in TC-100 medium supplemented with 10% (v / v) FCS and 0.26% bacto-tryptone. Tn-5 was cultured using serum-free ESF...

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Abstract

The invention discloses a method for producing recombinant human vascular endothelial cell growth factor VEGF by silkworm as a "biological factory". Using the hybrid baculovirus HyNPV of AcNPV and BmNPV as a vector, a recombinant virus containing human VEGF gene was constructed. Bombyx mori was used as the host of the recombinant baculovirus, and recombinant human VEGF with high biological activity was expressed at a high level. Solve the shortcomings of VEGF expression in Escherichia coli requiring complex renaturation operations, unstable plasmid transformants in yeast expression, and high production costs in mammalian cultured cells. Most of the recombinant human VEGF expressed in silkworm larvae is secreted into the blood In lymph, it is very conducive to the rapid extraction of protein. The experimental results prove that it is safe, practical and feasible to produce recombinant human VEGF using recombinant baculovirus and silkworm as a "biological factory". The mass production of recombinant VEGF has opened up broad prospects for the application of VEGF in the fields of medicine and therapy.

Description

technical field [0001] The invention relates to DNA recombination technology, protein purification and activity analysis, in particular to a method for silkworm as a "biological factory" to produce recombinant human VEGF. Background technique [0002] Human vascular endothelial growth factor VEGF is a specific mitogen and angiogenesis inducer of endothelial cells, as well as a mediator of vascular permeability. Human VEGF is a 165-amino acid polypeptide with a molecular weight of 19kDa and pI7.3. It is a glycoprotein containing two identical polypeptide chains connected by disulfide bonds. The unique biological activities of VEGF are specific to the vascular endothelium, including potent mitogenic and permeability-inducing properties. In addition, VEGF has been implicated in tumor angiogenesis, wound healing, and indirectly stimulates blood vessel formation at blocked arteries. Recent evidence suggests that VEGF is also important for embryonic vascularization and angiogene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/866C12N5/10
Inventor 吴小锋姚慧鹏曹翠平
Owner ZHEJIANG UNIV