Recombination and expression for non-antibiotic expression vector of rotavirus Vp6 gene and lactic acid bacteria
A rotavirus and expression vector technology, applied in viral peptides, genetic engineering, plant genetic improvement, etc., can solve problems such as environmental microecological damage
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[0080] 1. PCR amplification and cloning of the target gene
[0081] According to the gene sequence of (AF317123.) porcine rotavirus Wa strain Vp6 in GenBank, design a pair of PCR primers (synthesized by Dalian TaKaRa company) that amplifies this gene: P 1 : 5'-GTG GAGCTC ATGGAG GTT CTG TAC TCT TTA T-3';P 2 : 5'-GTG GGTACC TCA CTT AAT CAACAT GCT TCT-3' (the underlined sequences are SacI and KpnI restriction sites respectively). The total RNA of porcine rotavirus was extracted according to the method of Trizol kit; the Vp6 gene was amplified by RT-PCR using the total RNA as template and P1 and P2 as primers. The 50 μl reverse transcription reaction system and reaction conditions are as follows: take 10 μl mRNA, heat at 75°C for 5 minutes to denature the double-stranded RNA, and quickly place it on ice to cool, then add 10.0 μl of 5×AMV Buffer; 4.0 μl of dNTPs (10 mM each); RNasin 1.0μl; downstream primer 0.5μl; AMV (5Units / μl) 0.5μl; Olig(dT) 1.0μl; MgCl 2 (25mM) 4.0μl; ...
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