Prepn. and application tech. for pig interleukin-4 gene anti-diseases prepns.
A technology of interleukin and gene, applied in gene therapy, anti-infective drugs, allergic diseases, etc., can solve problems such as low efficiency and large DNA dose
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[0071] 1. Cloning of Tibetan pig IL-4 gene (TPIL-4 gene)
[0072] 10 mL of sterile blood was collected from the ear vein of healthy Tibetan pigs, and the peripheral blood lymphocytes of Tibetan pigs were separated, stimulated by adding 10 ug / ml Con A, and incubated in CO 2 Cultured in an incubator at 37°C for 72 hours, extracted the total RNA of Tibetan pigs in different periods (24, 48, 70 hours), and designed a pair of primers for amplifying the cDNA of the Tibetan pig IL-4 gene. The primer sequences are as follows:
[0073] Primer 1: CGG GAT CCC TAT TCA TGG GTC TCA CCT C
[0074] Primer 2: CGG GAT CCT CAG CTT CAA CAC TTT
[0075] Using total RNA as a template, add 5 μl of 10×RT-PCR reaction buffer, 25mM MgCl to the 50 μl reaction system 2 10μl, 10mM dNTP5μl, RNase Inhibitor (40units / μl) 1μl, AMV RNase XL (5units / uL) 1μl, AMV-Optimized Taq (5units / uL) 1μl, primer 1 and primer 2 each 20uM, total RNA 1-2μl, water to 50 μl. RT-PCR cycle parameters are: 50°C, 30min; 94°C, ...
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