甘薯褪绿矮化病毒RNase3蛋白的原核表达及抗血清制备

Prokaryotic expression of RNase3 of Sweet potato chlorotic stunt virus and preparation of its antiserum

以本实验室保存的重组质粒为模板,利用PCR方法扩增获得了甘薯褪绿矮化病毒SPCSV的RNase3基因,RNase3基因由690个核苷酸组成,编码229个氨基酸。将RNase3基因克隆到原核表达载体pET-28a(+),转化大肠杆菌BL21(DE3),经IPTG诱导,对诱导产物进行SDS-PAGE分析。结果表明,RNase3在大肠杆菌中能高效表达,融合蛋白分子量约为26.5kD。以表达的融合蛋白为抗原,免疫家兔,制备了SPCSV-RNase3的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清对RNase3融合蛋白的效价达10万倍。

With the recombinant plasmid stored in our laboratory as template,RNase3 gene of Sweet potato chlorotic stunt virus（SPCSV）was amplified by PCR.The full-length RNase3 gene consists of 690 nt and encodes 229 amino acid residues.The RNase3 gene was cloned into expression vector pET-28 a（＋）for over-expression in prokaryotic cells.The recombinant plasmid pET-RNase3 was transformed into Escherichia coli strain BL21（DE3）competent cells.SDS-PAGE result showed that specific fusion protein with the molecular weight of about 26.5 kD was produced after induction by IPTG,indicating that the RNase3 fusion protein was highly expressed in prokaryotic cells.The expressed protein was purified from SDS-PAGE and the antiserum against RNase3 protein was raised in rabbit.ACP-ELISA detection indicated that the titer of RNase3 antiserum was over 1∶100000 against RNase3 fusion protein.