禽网状内皮组织增殖病病毒PCR及RT-PCR检测方法的研究和p30主要抗原域的原核表达

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

根据MONAM.ALY发表的一对引物,5'-CATACTGGAGCCAATGGTGTAAAGGGCAGA-3'(上游引物),5'-AATGTTGTAGCGAAGTACT-3'(下游引物),上游引物位于REV-SNV毒株LTR序列上游237bp到267bp,下游引物在499bp到517bp之间,扩增产物总长为281bp。以REV-T株感染的鸡胚成纤维细胞DNA作为PCR扩增模板,进行PCR扩增,提取病毒RNA进行RT-PCR,这两种方法均获得了禽网状内皮增生症T株的部分LTR序列,将其克隆入pGEM-TEasy克隆载体中,经测序,证实为禽网状内皮增生症T株的部分LTR序列,初步建立了REV的PCR和RT-PCR检测方法。人工合成了REVp30的主要抗原域,用构建成功的表达禽网状内皮增生症p30主要抗原域的原核表达载体pET-p30,转化大肠杆菌BL21,挑取阳性克隆培养,IPTG诱导后裂解菌体作SDS-PAGE分析,出现大小约25Ku的表达产物即融合蛋白TrxA-p30主要抗原域,经Westernblot验证融合蛋白具有REV特异的反应原性。采用常规表达条件:LB培养基,37℃培养,待菌生长至OD600为0.4h～0.6h,用终浓度为0.2mmol/L～0.8mmol/L的IPTG进行诱导,于37℃振荡培养4h～5h,均能高效表达。本研究为建立REV抗体的ELISA检测方法打下基础。

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 237 bp to 267 bp to antisense primer of nucleotide 499 bp to 517 bp 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 Virus 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 major antigenic domain of p30. The domain was subcloned into enzyme sites EcoR Ⅰ , Hind Ⅲ of a prokaryotic expression vector pET32-α （＋）. The recombinant plasmid was named as pET-p30. The expression vector was transformed into E.coli strain BL-21. 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 25 Ku. 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 ℃,0.2 raM-0.8 mM 1PTG, incubating 4 h to 5 h after induction.The study is the basis of ELISA for detection of antibody against REV.