猪瘟弱毒C81株全基因组测序及分子结构预测

Complete Genome Sequencing and Molecular Structure Prediction of C81 Attenuated Strain of CSFV

参照已发表的猪瘟病毒弱毒株的序列,设计7对覆盖全长基因组的引物,通过RT-PCR从感染猪瘟病毒弱毒株的PK-15细胞中扩增得到7个cDNA片段,分别克隆到pMD18-T载体并测序,利用DNASIS软件获得猪瘟病毒C81株全基因组序列(GenBank:AY663656).C81株基因组全长12310nt,只有一个大的开放阅读框,编码3898个氨基酸的聚蛋白.序列分析表明,C81株开放阅读框与其它各毒株核苷酸和氨基酸序列的同源性变化较大,分别为84.4%～99.6%和91.6%～99.4%.同时,我们绘制了26株CSFV ORF的进化树,比较了CSFV 5'非翻译区核苷酸序列并推测其二级结构,发现不同毒株之间存在较大的差异,另外对C81株聚蛋白的功能域和三维结构进行了预测.

Seven pair of primers covering the whole genome of Classical swine fever virus（CSFV） attenuated strain were devised according to the published sequences . 7 cDNA fragments were amplified by RT-PCR from PK-15 cell infected by CSFV attenuated strain. The amplifid fragments were further sequenced after cloned into the pMD18-T vector. The whole genome sequence of C81-strain was assembled by DNASIS （GenBank： AY663656）. The genome is 12310 nucleotides in length and contains one large open reading frame encoding a polyprotein of 3898 amino acids. Sequence comparison with other CSFV strains revealed major differences in the open reading frame,84.4%-99.6% identities at the nucleotide level and 91.6%-99.4% identities at the amino acid level. 26 ORF phylogenetic trees of CSFV strain were also protracted. The nucleotide sequence and secondary structure of CSFV 5＇-untranslated region （5＇-UTR） were compared and deduced, and found the major difference exists among diverse virus strains. Furthermore, functional domains and 3-D structure of CSFV/C81 polyprotein were predicted.