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Method for producing recombinant canine interferon alpha from cultivated silkworm

A technology of alpha interferon and silkworm, applied in the field of DNA recombination technology, protein purification and activity analysis, to achieve high biological activity and facilitate rapid extraction

Inactive Publication Date: 2009-03-04
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

Reported the canine α-interferon gene sequence and patented the use of this gene, but there are few domestic studies on this aspect

Method used

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Embodiment Construction

[0014] In recent years, the dog industry has developed rapidly in my country, and keeping dogs as pets has become a part of people's lives, but viral diseases seriously endanger the health and survival of dogs. In order to develop and develop canine recombinant interferon biological preparations, this application cloned and sequenced the α-interferon gene, and established a technology for expressing recombinant canine α-interferon using my country's characteristic resource insect silkworm, which is the preparation of recombinant canine α-interferon Application lays the groundwork.

[0015] 1. Materials: DNA 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 pBlueBacHisA, lipofectin and Ni-NTA resin are all products of Invitrogen Company of the United States. DNA sequencing kits were purchased from PE Applied Biosystems. Fetal calf serum FCS and...

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Abstract

The invention discloses a method for expressing recombinant canine Alpha interferon with silkworm. Silkworm baculovirus serves as a carrier and recombinant virus containing a canine Alpha interferon gene is structured. Silkworm serves as a host of the recombinant baculovirus, and the recombinant canine Alpha interferon containing biological activity is expressed. The method solves the defects that complicated renaturation operations are needed in expressing the canine Alpha interferon in colon bacillus, the plasmid transformants in yeast expression are unstable and the production cost of mammal culture cells is too high, most of the recombinant canine Alpha interferon expressed by silkworm larvae is secreted into haemolymph, which is very good for the quick extraction of protein. Experimental results show that the method for using the recombinant baculovirus to produce recombinant canine Alpha interferon with silkworm is feasible. The largely expression of the recombinant canine Alpha interferon opens up broad prospects for the application of the Alpha interferon to medical and curing fields.

Description

technical field [0001] The invention relates to DNA recombination technology, protein purification and activity analysis, in particular to a method for producing recombinant canine alpha interferon by silkworm. Background technique [0002] Interferon (Interferon, IFN) is a kind of secreted glycoprotein produced by vertebrate cells. It has broad-spectrum antiviral and immune response enhancing effects, and its main activity is antiviral. The whole gene of canine interferon alpha is 561 bases, encoding 187 amino acids, of which the first 23 amino acids are signal peptides, the last 164 amino acids are mature proteins, and the molecular weight of the protein is about 19kDa. In 1987, Hiummler et al first started the study of canine α-interferon gene. Reported the canine α-interferon gene sequence and patented the use of the gene, but there are few domestic studies in this area. Contents of the invention [0003] The purpose of the present invention is to provide a method fo...

Claims

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Application Information

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IPC IPC(8): C12N15/21C12N15/866C12N7/01C07K14/56
Inventor 相兴伟吴小锋陈琳
Owner ZHEJIANG UNIV
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