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.
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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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