简化cDNA末端快速扩增技术测定基因Ⅲ、Ⅵb和Ⅶd型新城疫病毒基因组末端序列及分析

Analysis of leader and trailer sequence of genotype Ⅲ,Ⅵb and Ⅶd Newcastle disease virus determined by modified rapid amplification of cDNA ends (RACE) strategy

【目的】简化cDNA末端快速扩增技术(Rapid amplification of cDNAends,RACE)流程,测定基因Ⅲ、Ⅵb和Ⅶd型新城疫病毒(Newcastle disease virus,NDV)基因组两侧末端序列,并对NDV的leader和trailer进行分析。【方法】利用T4 RNA连接酶将特定寡聚核苷酸片段的连接于病毒基因组RNA和cDNA,再利用RT-PCR或PCR方法对病毒基因组的末端进行快速的扩增。【结果】建立一套操作简单、低成本、可重复性高的RACE方法,测定了三种基因型5株NDV3’末端leader和5’末端trailer序列比对分析。【结论】本实验测定的鹅源VII型毒株JS/7/05/Ch基因组的15,184 nt由一个T变为了C,5’端trailer与3’端leader的连续互补序列由8 nt变为12 nt,而其它4株基因Ⅲ型和VI型NDV均未发现该突变。通过RNA的二级结构分析,NDV基因组和反向基因组RNA的3’末端形成一个发卡结构。JS/7/05/Ch等3株NDV U→C(T→C)的突变位于发卡环上,不影响二级结构的形成,发卡环的RNA序列突变为3’-UCUC-5’,与基因组3’端发卡环的3’-UCUUA-5’相似,推测可能影响了基因组RNA的复制速度。

[Objective] The purpose of this research is to establish a simple rapid amplification of cDNA ends （RACE） strategy for direct mapping of the 3＇ end and 5＇ end of the genomic RNA of Newcastle disease virus （NDV）, and to analyze the leader and trailer sequence of NDV strains belonging to different genotypes. [Methods ] Classic RNA Ligase Mediated Rapid Amplification of cDNA Ends （RLM-RACE） was specifically modified for mapping both ends of the NDV genome. 3＇ -RACE was carried out by genomic RNA ligation with 5＇ end phosphated adaptor CL ＋ , and the 5＇ end was obtained by first strand cDNA with adaptor CL ＋ . [ Results] A modified RLM-RACE strategy was established in this paper, which proved simple, low-cost, repetitive and could be specifically used to map genome ends of NDV. By using this method, the leader and trailer sequence of 5 NDV strains, termed JS/5/05/Go, JS/07/04/Pi, JS/07/16/Pi, JS/7/05/Ch and JS/9/05/Go, belonging to genotype Ⅲ, Ⅵ and Ⅶ was determined, respectively. [ Conclusionl The initial 8nt at the 3＇ and 5＇ ends of the genome of genotype Ⅰ-Ⅵ NDV strains were complementary, whereas, the complementary sequences of strain JS/5/05/Go were up to 9 nt due to a mutation from T to C at the 9th nt in the 5＇ end. The 3＇ end of NDV genomic and anti-genomic RNA was predicted to form a potential hairpin structure. The U→C（ T→C）mutation was located in the circle part of the hairpin in the 5＇ end of anti-genomic RNA, and had no visible influence on the formation of RNA secondary structure. However, the sequence of the circle part of the hairpin was changed from 3＇-UUUC-5＇ to 3＇-UCUC-5＇ , more similar to the 3＇-UCUUA-5＇ in the hairpin of genomic RNA.