Ribosomal deactivated protein gene
A ribosome and gene technology, applied in genetic engineering, plant genetic improvement, sugar derivatives, etc.
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[0044] Specific embodiments of the present invention: use RA39 gene to obtain ribosome inactivating protein with enzymatic activity
[0045] 1. Acquisition of RA39p protein coding region
[0046] According to SEQ ID NO.1, synthetic gene-specific primer P5: 5'-GAT CACCAGCTTAGCCGTC-3' (nucleotides: 33-49) and P6: 5'-GATC ACACCTTGCATTAATACAACCA-3' (nucleotides: 935-955). An EcoRI restriction site was added to the 5' end of P5, and a SalI restriction site was added to the 5' end of P6 (shown in square brackets). The coding region (ORF) of the gene was amplified by RT-PCR.
[0047] The synthesis conditions of the first-strand cDNA are: 5 μg RNA and 0.5 μg oligo-dT 15 Mix the primers, add sterile water to make the volume of the mixture reach 12 μl, incubate at 70°C for 10 minutes, transfer to an ice bath, add 4 μl 5X first strand buffer, 1 μl 10mM dNTPs, 2ul 0.1mM DTT, preheat at 42°C for 2 minutes , add 1 μl SuperScript II RNaseH - Reverse transcriptase (200U / μl, GIBCO-BRL,...
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