Siberian tiger alpha interferon and coding gene and application thereof
A technology of alpha interferon and Siberian tiger, applied in the direction of interferon, application, cytokine/lymphokine/interferon, etc., can solve the problem that the treatment effect of interferon virus disease is not very significant
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Embodiment 1
[0054] Example 1 Amplification of Amur tiger IFN-α family gene sequence
[0055] 1. According to the simple and rapid extraction method of whole blood genomic DNA, extract genomic DNA from the blood of Siberian tigers;
[0056] 2. Using Amur tiger genomic DNA as a template, primers mtIFN-αS and mtIFN-αA as primers, PCR amplified IFN-α gene, wherein the nucleotide sequence of primer mtIFN-αS is: 5'-GATCCCCAATGGCGCTGCCCT-3', primer The nucleotide sequence of mtIFN-αA is: 5'-TCCATGTTGAACAGGTCTCATTTCT-3'; the reaction system and reaction conditions are as follows:
[0057] PCR reaction system:
[0058]
[0059] Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58.2°C for 30 s, extension at 72°C for 50 s, 30 cycles; final extension at 72°C for 10 min, and termination at 4°C. The results of electrophoresis of PCR products on 1% agarose gel were as follows: figure 1 shown.
[0060] The target fragment was recovered, and the p...
Embodiment 2
[0064] Example 2 Prokaryotic expression of Siberian tiger IFN-α family genes
[0065] In the process of prokaryotic expression, the signal peptide part of the Siberian tiger IFN-α family should be removed, so the primers TIFN-αS and TIFN-αA for expressing the mature peptide of the Siberian tiger IFN-α family should be designed first, TIFN-αS: 5'-ACG GGATCC TGTGACCTGCCTCAGACCCA-3' (underline: BamH I restriction site), TIFN-αA: 5'-CGT CTCGAG TCATTTCTCGCTCCTTAACCT-3' (underline: Xho I restriction site).
[0066] PCR amplifies the nucleotide sequence encoding the mature peptide of Amur tiger IFN-α family, and the reaction system is as follows:
[0067]
[0068] Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58.2°C for 30 s, extension at 72°C for 50 s, 30 cycles; final extension at 72°C for 10 min, and termination at 4°C. The results of electrophoresis of PCR products on 1% agarose gel were as follows: figure 2 shown. ...
Embodiment 3
[0074] Example 3 Functional verification of the antiviral activity of the recombinant Siberian tiger IFN-α family
[0075] 1. Prepare cat kidney cells at 37°C, 5% CO 2 Cultivate for 24 hours;
[0076] 2. Change the cell culture medium, insert 100 μL of 0.3 mg / ml recombinant Siberian tiger IFN-α family protein (prepared in Example 2 of the present invention) into the cat kidney cells simultaneously, and cultivate for 24 h;
[0077] 3. Change the cell culture medium, add 100 μL vesicular stomatitis virus (VSV), canine distemper virus (CDV), avian influenza virus (AIV) respectively at the same time, 100 pfu / well, cultivate for 24 hours, observe under a microscope, and test the virus The received inhibition (inhibition rate) is counted, and the results are as follows Figure 4-Figure 9 shown.
[0078] The results show that the Siberian tiger IFN-α family protein prepared by the present invention has a common conserved domain in its structure, i.e. the IFabd domain (its amino ac...
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