摘要
采用异硫氰酸胍 /酚 /氯仿抽提一步法 ,提取新城疫病毒 ( NDV) F4 8E9株及 2个东北地区分离株 DB3、DB5基因组 RNA,并以其为模板经反转录合成 F基因 c DNA第 1链 ,再利用 PCR技术分段扩增出 F基因的c DNA。F4 8E9、DB3和 DB5株 F基因的 c DNA经酶切修饰后 ,插入 p UC1 9的多克隆位点中 ,转化感受态 E.coli DH5α。经相对分子质量比较、酶切分析及 PCR等鉴定方法筛选出 F4 8E9、DB3和 DB5株 F基因的阳性克隆。然后采用 Sanger′s双脱氧末端终止法对阳性重组子进行核苷酸序列测定 ,证实分别获得了 F4 8E9、DB3和 DB5株 F基因的全长 c DNA。利用 DNASIS及 MEGA分析软件 ,将上述 3个毒株的 F基因序列与基他 1 1株有代表性的 NDV的相应序列进行比较分析 ,并绘制了 1 4株 NDV F基因的系统发育进化树。结果表明 ,F4 8E9和 DB3株在核苷酸序列上相对独立于 Toyoda等所分的 A、B、C组 ,单独成为一组 ;DB5株与B1 Hitchner株同属 B组 ,说明它们具有较高的同源性。同时也证明我国有不同基因型的毒株在流行。从进化的角度来看 ,NDV各毒株间确实存在遗传变异 ,但
Virion RNA were abstracted from purified NDV F48E9, DB3 and DB5 strain, and used as templates for cDNA synthesis, the cDNA of F gene were amplified by PCR and ligated with the pUC19. The positive clones of F gene of NDV F48E9, DB3 and DB5 strain were identified by molecular weight, restriction endonucleases digestion and PCR. The positive clones were sequenced by Sanger′s method. Comparison and analysis of the nucleotide sequence and deduced amino acid sequence with that of other 11 strains previously published was performed by computer with DNASIS and MEGA software. The results showed that a high degree of functional and structural constraint exerted on the F glycoprotein. However, there were amino acid residues variations in coding region of f gene. Phylogenetic tree of the nucleotide sequence of NDV strains F gene was constructed by computer. The F48E9 and DB3 strains were found to be able to construct a new group independent from classic A,B,C groups, and the DB5 strain should belong to group B.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2000年第2期117-120,共4页
Chinese Journal of Veterinary Science
基金
国家"八五"攀登计划 B项目 !( 85 -4 4 -0 1-4 4 )
国家自然科学基金重大项目! ( 3 9893 2 90 -4 )
