Procambarus clarkia anti-lipopolysaccharide factor gALF1 gene, coded gALF1 protein thereof and application thereof
A technology for resisting lipopolysaccharide factor and Crayfish is applied in the directions of application, genetic engineering, plant genetic improvement, etc., which can solve the problems of economic loss, death, and disease is not good, and achieves the level of inhibiting replication and strong inhibitory effect. Effect
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[0059] Preparation of gALF1 protein
[0060] ① RNA extraction and reverse transcription:
[0061] Three Procambarus clarkii were selected, blood was drawn and dissected to take tissue samples: hemolymph, heart, liver, gills, stomach, and intestine, and total RNA was obtained by TRIzol method. Nano-Drop 300 was used to detect the purity and concentration of RNA, and the quality of total RNA was analyzed by agarose gel electrophoresis using a Bio-Rad gel imager; cDNA was synthesized by reverse transcription according to the reverse transcription instructions.
[0062] ② ALF1 full-length coding region gene amplification:
[0063] upstream primer
[0064] Pc-ALF1-EcoR I: 5'(TACTCAGAATTCCAGGTCCTGGAGGGTCTG)3';
[0065] downstream primer
[0066] Pc-ALF1-Xhol I: 5'(TACTCA CTCGAGCTACCCATCAAGCCATGCCT) 3'.
[0067] The cDNA obtained by reverse transcription was used as a template for PCR amplification; a 25 μL PCR reaction system was established, and the reaction conditions were: p...
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