兔病毒性出血症病毒基因组3'端非编码区的分子克隆及生物信息学分析

采用3′RACE方法对兔病毒性出血症病毒(RHDV)JX／CHA／97株基因组3′端全序列进行了扩增、克隆和测序,并利用ONAstar和DNAman软件分析了RHDV JX／CHA／97株与各参比毒株3′NCR的序列同源性,用RNA structure软件对3′NCR的二级结构进行了预测和分析。结果表明,所扩增的目的基因片段长1500 bp,包括部分VP60基因序列、ORF2基因、3′端非编码区(3′Non Coding Region,3′NCR)和poly(A)尾巴,其中 3′NCR位于ORF2终止密码子TAG之后,长59 bp,poly(A)至少含有27个A;3′NCR序列具有较高的同源性 (93．5％-100％);3′NCR二级结构可以形成2个潜在的茎-环结构(SL1和SL2),其中SL2具有一定的保守性,其形状和形成位置与poly(A)尾巴的长度关系不大,但是SL1的结构和形成位置与poly(A)尾巴的存在与否密切相关,提示poly(A)尾巴与3′NCR高级结构的稳定性以及基因组复制有一定关系。

Cloning and analyzing of the secondary structure of the 3'noncoding region of RHDV

The authentic 3' end sequence of Rabbit hemorrhagic disease virus (RHDV) including part of VP60,VP12,3'NCR,and poly (A) tail,was amplified by 3'RACE,and the result showed 3'NCR was 59nt in length,and at least 27 As existed in the poly(A) tail. Compared with other reference strains,JX/ CHA/97 strain share higher nucleotide acid identity with the reference strains (93. 5%-100%). With the help of RNA structure soft ware,the secondary structure of RHDV was predicted and analysed. The result showed that there were two stem-loops (SL) in the region,which may be necessary for viral replication. Besides, it was found that the formation of SL1 was closely related to the presence of poly (A) tail, which implied that poly (A) tail may be related to the stability of the second structure of 3' NCR,and to the replication of viral genom.