摘要
采用RT-PCR技术扩增了HCV 10个野毒株、4批不同厂家生产的兔化弱毒疫苗株和1个标准SM株Erns基因的cDNA片段并对其进行了序列测定。应用DNAstar序列分析软件对所测的15个HCV毒株与国内外6个参考毒株Alfort、Ald、Brescia、Gpe、C、CW株的相应片段进行了同源性比较分析。15株HCV Erns基因长度均为696 bp,其核苷酸及氨基酸的同源性分别为83.0%~100.0%和87.9%~100.0%。系统发生分析可将这些毒株分为Ⅰ和Ⅱ两大基因群及4个亚群,Erns基因与E2、NS5B基因在系统发生分析中具有较高的一致性。国内4批不同厂家生产的兔化弱毒疫苗相对稳定,仅1~2个氨基酸残基具有差异。通过对10个流行株与SM株Erns基因的比较分析,发现我国HCV具有复杂性、多样性和相对的遗传稳定性;所测15株HCV Erns基因具有RNase活性的2个区域SLHGIWPG(或E)(28~36位)和EWNKHGWC(75~82位)十分保守,但疫苗毒SLHGIWPE基序中的E替换为G,而基序中位于30和79位的H(组氨酸)为RNase的催化活性关键氨基酸残基,高度保守,15个HCV Erns基因和6株参考株没有一株发生变异。本研究为开展Erns基因的生物学活性、结构与功能等方面的研究奠定了基础。
The cDNA of the Erns gene were molecularly cloned from viral particles of 10 HCV field isolates, 4 vaccinestrains and 1 standard strain Shimen(SM) using the reverse transcription-polymerase chain reaction (RT-PCR) method. Theresulted products (696 bp for all the 15 isolates, vaccine strains or standard strain) were subjected to DNA sequenceanalysis and compared with those of 6 reference strains Alfort, Ald, Brescia, Gpe, C and CW by using DNAstar software.The result showed that the nucleotide sequence and the amino acid sequence of the 21 HCV strains shared an 83.0%-100%and an 87.9%-100% homology respectively. The 21 HCV strains were divided into 2 major groups, HCV group Ⅰbeingrepresented by the strain Brescia and HCV group Ⅱ by Alfort, and four subgroups. Through analysis of the Erns gene of the10 field isolates and the SM strain, HCV was found to be of complexity, diversity and stability. The two motifs of Erns RNaseactivity,SLHGIWPG(or E) (28-36 sites) and EWNKHGWC (75-82 sites) were highly conserved, there being only onemutation from E to G in the domain SLHGIWPG in the 5 vaccine strains, no mutation being found in the key amino acidresidue H located in the 30 and 79 sites. Through comparison of our data and the published, it was found that the NS5B genewas the most conserved, Erns the second, and E2 the third.
出处
《中国农业科学》
CAS
CSCD
北大核心
2004年第3期446-452,共7页
Scientia Agricultura Sinica
基金
国家自然科学基金重大资助项目(39893290-1-2)
国家重点基础研究发展规划项目(G1999011903)
