鸡新城疫病毒(青岛株)分离鉴定及HN基因的克隆、序列分析与同源性比较

Isolation and Identification of NDV Strain Qingdao and NDV HN Gene in Homology Comparison with NDV Strains Offshore

本研究对新城疫病毒(NDV)青岛株进行生物学特性鉴定 ,结果表明 :该病毒的MDT为57.9h ,ICPI为1.65,IVPI为2.75 ;血凝解脱为快速解脱型 ,56℃水浴处理5min后失去血凝活性。应用RT-PCR一次性成功地扩增出该病毒的HN基因 ,将其克隆入载体pBluescriptⅡKS( -)中 ,经测序证实所扩增HN基因长度为1713bp,编码571个氨基酸。该HN基因的氨基酸序列中有6个糖基化位点 ,13个半胱氨酸残基 ,与国外发表的同类型NDV毒株HN序列中的数目和位置完全相符。同时与国外9株NDV的HN基因序列相比较 ,核苷酸和氨基酸的同源性分别为88.4%～92.9%和90.0%～94.2 %。系统发育进化树分析表明 ,NDV青岛株与Italien强病毒株亲缘关系最近 ,进一步证明 ,NDV青岛株为强毒株。

The study of biological character revealed that the mean death time(MDT) of chicken embryos was 57.9h and introcerebral pathogenicity index (ICPI) in 1-day-old chicken was 1.65 and intravenous pathogenicity (IVPI) in 6-week-old chicken was 2.75. The genomic RNA of the NDV strain Qingdao was extracted. The HN gene of the virus has been amplified from viral genomic RNA by RT-PCR. Then it was cleaved with BamHⅠand XbaⅠ, and directly cloned into vector pBluescriptⅡKS(-). By the sequence analysis, the HN gene amplified by RT-PCR was 1792bp long as expected. It included the complete encoding region of HN gene of NDV strain Qingdao. The nucleotide sequence of this HN gene encoding region was 1713bp and encoded a protein 571 amino acids. For the amino acid sequence, there are 6 glycosylation site and 13 cysteine resides. Compared with the published 9 NDV strains, the homology of the nucleotide sequence is 88.4%～92.9%, the homology of the deduced amino acid sequence is 90.0%～94.2%. Phylogenetic tree showed the more relationship between the NDV strain Qingdao and Italien strain. The result showed the NDV strain Qingdao was virulent.