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以大麦黄矮病毒 ( BYDV) GAV株系的 RNA为模板 ,通过 RT- PCR扩增获得通读蛋白 ( readthrough protein RTP)基因的目的片段。将其重组到 p GEM- 7zf( + )并转化 JM1 0 9得到了含有完整 RTP基因的重组子。采用双脱氧终止法进行序列分析。结果表明 ,RTP基因为1 377nt,与文献〔1〕报道的血清学较密切的 BYDV MAV- PS1相比 ,核苷酸和氨基酸的同源性分别为 87.4%和 87.1 %。将此 RTP基因克隆到原核表达质粒 p ET- 5a上 ,在大肠杆菌 BL2 1( DE3)中用 IPTG诱导表达 ,利用分离包含体的方法结合 SDS- PAGE电泳 ,得到了纯化的分子量为 66ku的非融合蛋白
The target fragment of readthrough protein RTP gene was amplified by RT-PCR using the RNA of BYDV GAV strain as a template. Recombinants containing the complete RTP gene were obtained by recombination into p GEM-7zf (+) and transformation of JM109. Sequence analysis was performed by dideoxy termination method. The results showed that the RTP gene was 1 377nt. Compared with the serologically isolated BYDV MAV-PS1 reported in [1], the nucleotide and amino acid homologies were 87.4% and 87.1%, respectively. The RTP gene was cloned into the prokaryotic expression plasmid p ET-5a and induced by IPTG in E. coli BL21 (DE3). The inclusion body was separated by SDS-PAGE electrophoresis and the purified molecular weight was 66 ku Fusion protein