Chicken gamma-interferon expression gene modification method and gene obtained by same
A technology for expressing genes and interferon gamma, which is applied in the field of genetic engineering, can solve the problem of low yield of interferon gamma in chickens, and achieve the effects of improving expression efficiency, short production cycle and low cost
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Embodiment 1
[0031] This example describes the method for obtaining the natural chicken interferon gamma (ChIFN-γ-N) gene and the optimized chicken interferon gamma (ChIFN-γ-O) gene provided by the present invention.
[0032] According to the mRNA sequence of FJ788637 in GeneBank, after removing the signal peptide sequence, it is the nucleotide sequence of the natural chicken gamma interferon (ChIFN-γ-N) gene provided by the present invention (see SEQ ID NO1).
[0033] Through the analysis of E. coli codon analysis software, and according to the codon preference of E. coli, the natural chicken gamma interferon (ChIFN-γ-N) gene was replaced with rare codons and regulated GC content optimization; natural gamma interferon contains two cysteines at the C-terminus, the present invention found that these two cysteines have no effect on the biological activity of interferon. So optimize the gene so that it mutates into a serine with similar properties. Through the above optimization, the optimiz...
Embodiment 2
[0035] This example describes the construction of recombinant plasmids and engineering bacteria containing the ChIFN-γ-O gene.
[0036] 1. Amplification of ChIFN-γ-O gene
[0037] According to the above ChIFN-γ-O gene sequence, primers were designed using Primer Premier software, and at the same time, Nco I restriction site was introduced at the 5' end of the primers, and a Hind III restriction site was introduced at the 3' end:
[0038] Upstream primer (F1): 5'- CCATGG GTCATACCGCAAGCAGCCTG-3' (the underlined part is the Nco I restriction site);
[0039] Downstream primer (R1): 5'- AAGCTT TTAGCTATTGCTACGACGC-3' (the underlined part is the Hind III restriction site).
[0040] The synthetic ChIFN-γ-O gene sequence was used as a template, and F1 and R1 were used as primers for PCR amplification. The reaction system contained 1 μg of template, 1 μM of upstream and downstream primers, and the total reaction system was 50 μL; the reaction conditions were: 94 ° C, 5 min ; 94°C, 3...
Embodiment 3
[0049] This example describes the induced expression of the recombinant engineered bacteria BL21(DE3)-ChIFN-γ-pET-28a(+), and the purification of the expressed recombinant chicken interferon-γ (ChIFN-γ-O).
[0050] 1. Induced expression of recombinant engineering bacteria BL21(DE3)-ChIFN-γ-pET-28a(+)
[0051] 1. Inoculate the recombinant engineered bacteria BL21(DE3)-ChIFN-γ-pET-28a(+) in 100mL containing ampicillin-resistant (Kan + ) in liquid LB medium, cultured on a shaker at 37°C and 200 rpm until OD 600 When the value is 0.8, add the inducer IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration 0.5mM) 180rpm to continue the induction culture, and the induction time is 5 hours; Except for the inducer IPTG, other operations were the same.
[0052] 2. Take 5 mL of the induced and uninduced fermentation broths respectively, and centrifuge at 8000-10000 rpm for 20 minutes to collect the bacteria. Then add 500 μL Tris-HCl (pH 7.9) to resuspend the bacteria, after ul...
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