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Construction method and application of canine caifn-α gene expression plasmid

A technology for expressing plasmids and constructing methods, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., and can solve complex renaturation operations, silkworm larva expression that cannot be produced every year, and yeast expression plasmid transformants that are unstable and other issues to achieve a good expression effect

Inactive Publication Date: 2011-12-14
ZHEJIANG UNIV
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Problems solved by technology

Regarding the expression of canine CaIFN-α, methods such as Escherichia coli, yeast, mammalian cultured cells and silkworm larvae have been used, but there are the following shortcomings: expression in Escherichia coli requires complex renaturation operations, and plasmid transformants in yeast expression Unstable, the cost of mammalian cultured cell production is too high, and the expression of silkworm larvae is limited by seasons and cannot be produced year-round

Method used

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  • Construction method and application of canine caifn-α gene expression plasmid
  • Construction method and application of canine caifn-α gene expression plasmid

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Embodiment 1

[0025] 1), construction of shuttle vector plasmid:

[0026] Plasmid pUE30 was extracted from radiation-resistant bacteria (Deinococcus radiopugnance ATCC19172) and digested with SphI; then digested with SphI containing the E. coli vector pUC19 replication initiation domain, the active Amp resistance gene in E. Plasmid pKatCAT (Funayama et al., Mutat. Res., 435:151-161, 1999) with an active chloramphenicol resistance gene in R1 was mixed with the above Sph I digest of pUE30 and ligated with DNA ligase. The above linker was transformed into Escherichia coli JM109 strain by electroporation. A strain having the pUE30 and pKatCAT-linked plasmids was selected from the Amp-resistant transformants to obtain the shuttle vector plasmid pZT29.

[0027] 2), the cloning of CaIFN-α gene:

[0028] Genomic DNA extracted from the blood lymphocytes of local domestic dogs in Zhejiang Province was used as a template, and the base sequences shown in SEQ ID NO: 1 (i.e. sequence 1) and SEQ ID NO: ...

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Abstract

The present invention is based on the shuttle vector between radioresistant bacteria and Escherichia coli, and is recombined with the active groEL promoter and canine CaIFN-α gene fragment derived from the radioresistant bacteria Deinococcus radiodurans to construct a plasmid with CaIFN-α expression activity. The radioresistant bacterium Deinococcus grandis was used as the host, induced by -ray irradiation, etc., to efficiently express and produce canine CaIFN-α with biological activity. The present invention better solves the complex renaturation operation required for the expression of canine CaIFN-α in Escherichia coli, the instability of plasmid transformants in yeast expression, the high cost of expression and production in mammalian cultured cells, and the need for expression in silkworm larvae. Due to seasonal restrictions, the disadvantage of not being able to produce on an annual basis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a gene plasmid and its construction and expression method, that is, the construction of the canine CaIFN-α expression plasmid and the method for expressing the canine CaIFN-α in radiation-resistant bacteria. Background technique [0002] In recent years, dogs, as companion animals, police dogs and rescue dogs, have become more and more important in human life. However, various viral diseases plague China's dog breeding industry. On the one hand, there is no specific therapeutic drug for virological infectious diseases; on the other hand, the occurrence of zoonotic pathogens such as rabies and canine parvovirus It has brought great harm to human beings and has attracted the attention of the whole society. Therefore, from the perspective of veterinary medicine and human medicine, comprehensive control of canine viral diseases requires safe, effective, and less side effect drugs, and can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/66C12P21/02C12R1/01
Inventor 屠振力
Owner ZHEJIANG UNIV
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