Detection Of Reticuloendotheliosis Virus Infection Using PCR Or RT-PCR And Expression Of The Major Antigenic Domain On Protein P30 Of Reticuloendotheliosis Virus In E.coli

Based on a pair of primers publicated by MONA M.ALY (Sense primer 5'- CAT ACT GGA GCC AAT GGT GTA AAG GGC AGA-3',antisense primer 5'-AAT GTT GTA GCG AAG TAC T-3'), PCR extends from sense primer of nucleotide 237nt to 267nt to antisense primer of nucleotide 499nt to 517nt of the long terminal repeat of SNV. A fragment of 281 bp of LTR gene was amplified by Polymerase Chain Reaction (PCR) from the genome of CEF infected with Reticuloendotheliosis Viruse T strain (REV-T) or RT-PCR from viral RNA and cloned into plasmid pGEM-T Easy. The result of PCR or RT-PCR showed that the designed fragment was amplified with expected molecular weight and named as LTR.Two enzyme sites EcoR I ,Hind III were introduced at 5'terminal and 3'terminal of p30.The p30 was subcloned into enzyme sites EcoR I ,Hind III of a prokaryotic expression vector pET32-a(+). The recombinant plasmid was named as pET-p30.Expression vector of major antigenic domain on p30 protein was transformed into E. coli strain BL21.The host was named BL-p30. After inducing by IPTG, the expression production experiment showed that fused protein TrxA-p30 was expressed in BL-p30 with expected molecular weight 30 KD.The result of Western-blot showed that TrxA-p30 had the antigenic characteristic of REV.The usual conditions for induction of TrxA-P30 can get high expression :37℃, 0. 2mM-0.8mM IPTG, incubating 4-5hrs after induction. The study is the basis of ELISA for detection of antibody against REV.