摘要
设计18对PCR引物分片段扩增猪戊型肝炎病毒新疆分离株swCH25全基因,对两末端采用末端快速扩增方法(RACE)进行扩增,扩增产物克隆到pMD18-T载体并测序。用DNAstar软件进行序列同源性分析,PHYLIP软件绘制基因进化树,在全基因序列的基础上比较猪HEV与其它HEV基因型的差异和人源HEV的关系。结果显示swCH25全序列除去3′poly(A)尾,由7243个核苷酸组成,ORF1～3分别编码含1070,674和114个氨基酸的蛋白质。全基因序列与HEVⅠ、Ⅱ和Ⅲ型同源性73.5%～75.7%,与Ⅳ型的同源性为83.5%～89.8%,其中与Ⅳ型中国人源T1株最高,达89.8%。ORF1与Ⅰ～Ⅲ和Ⅳ型的核苷酸同源性分别为71.6%～74%和82.3%～89.4%,氨基酸同源性为80.7%～86.1%和94.3%～96.0%;ORF2与Ⅰ～Ⅲ和Ⅳ型的核苷酸同源性分别为78.4%～81.3%和87.1%～90.9%,氨基酸同源性为89.7%～93.8%和97.2%～98.2%;ORF3与Ⅰ～Ⅲ和Ⅳ型的核苷酸同源性分别为84.4%～87.3%和95.4%～96.8%,氨基酸同源性为74.8%～83.2%和93.8%～97.4%,基因进化树显示swCH25与基因Ⅳ型人源T1株最接近,在同一分支上。swCH25株是国内第一个测出全基因序列的猪戊型肝炎病毒。
To analyze the full length sequence of swCH25, isolated from a pig fecal sample in Xinjiang in 2001, eight overlapping fragments of swCH25 genomes were amplified with reverse transcription nested polymerase chain reaction （RT-nPCR） and the 5＇ and 3＇ ends were amplified with RACE. The PCR products were cloned into pMDI8-T Vector and sequenced. The result showed that its genome consisted of 7243 nt excluding the poly（A）tail, and contained three ORFs（ORFs1-3） that encoded proteins of 1707, 674 and 114aa. Sequence analysis revealed that the overall nucleotide sequence identity of swCH25 was 73.5%-75.7% and 83.5%-89.8% with HEV genotype Ⅰ-Ⅲ and Ⅳ, respectively. Compared with HEV genotypes Ⅰ-Ⅲ and Ⅳ, the ORF1 of swCH25 shared 71.6% 74% and 82.3%-89.4% sequence identity at nucleotide level and 80.7%-86.1% and 94.3% 96% identity at amino acid level, the ORF2 shared 78.4% 81.3% and 87.1%-90.9% sequence identity at nucleotide level and 89.7%-93.8% and 97.2%-98.2% identity at amino acid level and the ORF3 shared 84.4%-87.3% and 95.4-96.8% sequence identity at nucleotide level and 74.8% 83.2% and 93.8%-97.4% identity at amino acid level. The phylogenetic tree constructed based on the full genomic sequence confirmed that swCH25 belong to genotype Ⅳ and were most closely related to T1, a prototype human HEV strain of genotype Ⅳ isolated from patients with acute hepatitis in China. This finding further supports the notion that the hepatitis E is a zoonosis.
出处
《中国农业科学》
CAS
CSCD
北大核心
2005年第8期1669-1674,共6页
Scientia Agricultura Sinica
