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Canine recombinant interferon alpha and preparation method

A technology for recombinant interferon and canine interferon, applied in the directions of interferon, botanical equipment and methods, biochemical equipment and methods, etc.

Inactive Publication Date: 2012-01-18
CHANGCHUN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xia Chun et al. used PCR technology to amplify and clone the interferon gene alphaIFN of Labrador Retriever and German Shepherd and sequenced [Xia Chun, Wang Ming, Xia Zhaofei. Labrador and German Shepherd Interferon alpha Gene cloning and sequencing. Journal of Agricultural Biotechnology 1999, 7 (3)] but no expression, application research

Method used

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  • Canine recombinant interferon alpha and preparation method
  • Canine recombinant interferon alpha and preparation method
  • Canine recombinant interferon alpha and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] 1) Acquisition of the gene encoding canine recombinant interferon α

[0063] Referring to the CaIFN-α gene sequence, synthesize the target gene fragment, the gene sequence is as follows:

[0064] ATG TGC CAT CTG CCG GAC ACC CAT GGC CTG CGT AAT TGG CGT GTG CTG ACG CTG CTG GGC 60

[0065] CAG ATG CGT CGT CTG AGC GCG GGT AGC TGC GAC CAT TAT ACC AAC GAT TTT GCG TTC CCG 120

[0066] AAA GAG CTG TTT GAT GGT CAA CGT CTG CAA GAA GCC CAG GCG CTG AGC GTG GTG CAT GTG 180

[0067] ATG ACC CAG AAA GTG TTT CAC CTG TTT TGC CCG GAT ACG AGC AGC GCG CCA TGG AAT ATG 240

[0068] ACC CTG CTG GAA GAA CTG TGC AGC GGC CTG AGC GAA CAG CTG GAT GAT CTG GAA GCG TGC 300

[0069] CCA CTG CAG GAA GCC GGT CTG GCG GAA ACC CCG CTG ATG CAT GAA GAT AGC ACC CTG CGC 360

[0070] ACC TAC TTT CAG CGC ATT AGC CTG TAT CTG CAG GAC CGT AAC CAT AGC CCG TGC GCG TGG 420

[0071] GAA ATG GTG CGT GCG GAA ATT GGC CGT AGC TTT TTC AGC AGC ACG ATC CTG CAA GAG CGT 480

[0072] ATT CGT CGT GGC AAA TAA 498

[0073] I...

Embodiment 2

[0101] 1) Acquisition of recombinant gene encoding canine interferon α

[0102] Method Referring to Example 1 to obtain the canine recombinant interferon alpha gene

[0103] 2) Acquisition of E.coli BL21 engineering bacteria

[0104] The method refers to the acquisition of the E. coli BL21 engineering bacteria that obtained the recombinant gene through transformation in Example 1.

[0105] 3) Fermentation expression

[0106] Inoculate the engineered bacteria in 200ml LB culture medium with 1% inoculum amount, shake and cultivate overnight at 30°C; inoculate with 5% in 1L LB culture medium, transfer to the second generation at 30°C and cultivate until the OD value is 0.6, then inoculate with 10% Cultivate in a 50L fermenter at 30°C until the OD is 1-2, quickly raise the temperature to 42°C, express the bacteria, culture for 4-5 hours, centrifuge at 4000r / min for 30min, and collect 270g of the expressed bacteria; the expression level of the target protein is about Accounting ...

Embodiment 3

[0126] 1) Acquisition of recombinant gene encoding canine interferon α

[0127] Method Referring to Example 1 to obtain the canine recombinant interferon alpha gene

[0128] 2) Acquisition of E.coli BL21 engineering bacteria

[0129] The method refers to the acquisition of the E. coli BL21 engineering bacteria that obtained the recombinant gene through transformation in Example 1.

[0130] 3) Fermentation expression

[0131] Inoculate the engineered bacteria in 200ml LB culture medium with 1% inoculum amount, shake and cultivate overnight at 30°C; inoculate with 5% in 1L LB culture medium, transfer to the second generation at 30°C and cultivate until the OD value is 0.6, then inoculate with 10% Cultivate in a 100L fermenter at 30°C until the OD is 1-2, then quickly raise the temperature to 42°C, express the bacteria, culture for 4-5 hours, centrifuge at 4000r / min for 30min, collect and express 600g; 25% of total body protein (see Figure 7 );

[0132] 4) Bacterial fragmen...

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Abstract

The invention discloses canine recombinant interferon alpha, and also provides a preparation method of the canine recombinant interferon alpha. Recombinant genes of canine interferon alpha are expressed in escherichia coli, and canine interferon alpha with a purity of more than 98% is obtained through processes of renaturation and purification. The biological activity is determined by a MDCK (canine kidney cell)-VSV (vesicular stomatitis virus) virus system, and the specific activity reaches 2-3*107 IU / mg.

Description

technical field [0001] The invention discloses a canine recombinant interferon alpha, and also provides a preparation method of the canine recombinant interferon alpha, which is a genetic engineering method for producing the recombinant canine interferon and belongs to the field of biological products. Background technique [0002] Along with domestic working dogs (military dogs, police dogs, experimental dogs, etc.), meat dogs and pet dogs, etc., the amount of dogs raised has increased significantly, and viral diseases have become the direct cause of threats to the lives of dogs. Close contact between humans and dogs greatly increases the chances of humans being infected by the virus. For example, rabies virus, canine parvovirus and canine distemper virus will pose a great threat to human health and have attracted the attention of the whole society. In veterinary clinics, due to reasons such as the use, storage, and transportation of vaccines, immunization often fails, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C12N15/21C12N15/70C07K1/36C07K1/34C07K1/20C07K1/16C12R1/19
Inventor 殷玉和李莹莹刘新涛
Owner CHANGCHUN UNIV OF TECH
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