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目的对保存的WJBC株马雅罗病毒(MAYV)基因组序列进行测定,阐明其与已报道毒株的关系及追溯其可能来源。方法将MAYV基因组编码区分10段进行RT-PCR扩增,扩增产物直接进行测序,非编码区采用RACE法进行扩增,扩增产物纯化并连接pGEM-T easy载体后转化大肠杆菌DH5α感受态细胞,挑取阳性克隆鉴定后进行测序,用DNAStar软件将测序结果拼接得到基因组序列。下载MAYV核苷酸序列,利用MEGA5.0软件构建系统进化发生树。结果与结论 WJBC株MAYV基因组核苷酸(nt)共11 429 nt,编码3679个氨基酸,其中5’端的2/3基因组编码4种非结构蛋白nsP1,nsP2,nsP3,nsP4;3’端的1/3基因组编码5种结构蛋白C、E3、E2、6K和E1蛋白。结构基因和非结构基因之间有26 nt的连接区为非翻译区。病毒基因组5’端和3’端分别有76、290 nt的非编码区。进化树结果显示,WJBC株马雅罗病毒与TRVL4675株MAYV亲缘性最高。
OBJECTIVE: To determine the genomic sequence of WJBC strain Mayja virus (MAYV) and to elucidate its relationship with reported strains and to trace its possible origin. Methods The MAYV genome was divided into 10 segments for RT-PCR amplification. The amplified product was directly sequenced and the non-coding region was amplified by RACE. The amplified product was purified and ligated with pGEM-T easy vector and then transformed into competent E. coli DH5α The cells were picked and positive clones were identified and sequenced. DNAStar software was used to stitch the sequencing results to obtain the genomic sequence. Download MAYV nucleotide sequence, the use of MEGA5.0 software to build phylogenetic tree. RESULTS AND CONCLUSION: The nucleotide sequence of MAYV genome in WJBC strain was 11 429 nt, encoding 3679 amino acids. The 2/3 genome at the 5 ’end encodes four nonstructural proteins, nsP1, nsP2, nsP3 and nsP4. The 1 / 3 genome encodes five structural proteins C, E3, E2, 6K and E1 proteins. The 26 nt junction between the structural and nonstructural genes is the untranslated region. There are 76,290 nt non-coding regions in the 5 ’end and 3’ end of the viral genome respectively. The phylogenetic tree showed that WJBC strain Mayarrow virus had the highest affinity with MAYV of TRVL4675 strain.