SEN病毒中国分离株基因克隆及序列分析

目的：测定SENV-D亚型中国分离株的基因组序列，并对其进行初步分析。方法：根据国外已发表的SEN病毒基因序列设计特异性引物，应用套式PCR方法从一份非甲-非戊型(non-A-E)肝炎患者血清中，分段扩增了包括SENV-D型所有编码区(ORFs)长3175bp的DNA片段，将其克隆到T载体后测序。结果：测序结果经BLAST软件分析，与国外发表的3株D型SEN病毒SENV-D(AX025730)，SENV-D(AB059352)，TTV(AB028668)的核苷酸序列同源性分别为90％，88％和91％；与2株D型SEN病毒SENV-D(AX0Z5730)，TTV(AB028668)0RF1所编码蛋白的氨基酸同源性分别为93％和92％。ORF1蛋白中含有Rep蛋白(在病毒复制中起作用的一种蛋白)的两个保守基序，此外还有一个保守的ATP／GTP结合基序(P-loop)以及若干个高度保守的蛋白激酶磷酸化位点。结论：测定得到了SENV-D亚型中国分离株的基因组序列，有助于其检测方法及致病性的研究，并为研究SEN病毒的进化地位提供方便。

Cloning and sequencing of the genes of a Chinese SEN virus D subtype isolate

Objective:To sequence and analyse the Chinese strain of SENV-D genome for understanding its molecular characteristics.Methods:According to the published nucleotide sequences of SEN virus genome,specific primers were designed and synthesized.From the serum of a Chinese patient with non-A-E hepatitis,two long fragments (totally 3?175?bp) spanning the complete coding region of SENV-D variant gene were amplified by semi-nested PCR.The amplified fragments were cloned and sequenced.Results:The nucleotide sequence homology of this Chinese strain with the 3 published strains SENV-D(AX025730),SENV-D(AB059352)and TTV(AB028668) were 90%,88% and 91% respectively and the amino acid sequence homology of the protein encoded by ORF1 with 2 strains the SENV-D(AX025730) and TTV(AB028668) were 93% and 92%,respectively.The protein encoded by ORF1 had two conserved motifs which were conserved in putative replication-associated proteins(Rep proteins),one conserved ATP/GTP-binding motif and several highly conserved protein kinase phosphorylation sites.Conclusions:The genome sequence of Chinese strain might be used in studying its pathogenesis and phylogenesis.