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Method for preparing recombinant vEGF using domestic silkworm as biological factory

A silkworm and biological technology, which is applied in the fields of protein purification and activity analysis, and DNA recombination technology, can solve the problems of high cost of mammalian cells, unstable yeast plasmid transformants, etc. Effect

Inactive Publication Date: 2005-03-16
ZHEJIANG UNIV
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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 blood vessel endothelial cell growth factor VEGF by employing domestic silkworm as bio-factory. The invention uses baculovirus HyNPV hybridized by AcNPV and BmNPV as vector, constructs recombinant virus containing human VEGF gene, expresses highly recombinant human VEGF with biological activity while using domestic silkworm as baculoviral host, solves the shortcomings that it needs complicated operations in VEGF expression in escherichia coli, that plasmid transformer is not stable in yeast expression, and that the cost is high in mammal cell expression. recombinant human VEGFs expressed by domestic silkworm lava are mainly secreted into haemolymph, which favors the rapid protein extraction. Results shows that its is safe and practical that domestic silkworm is used as bio-factory and recombinant baculovirus as vector. Recombinant VEGF can be scale produced, which opens broad prospect in medical and therapeutical fields.

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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IPC IPC(8): C12N5/10C12N15/10C12N15/12C12N15/866
Inventor 吴小锋姚慧鹏曹翠平
Owner ZHEJIANG UNIV
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