鹅副粘病毒F蛋白基因的克隆和序列分析

鹅副粘病毒分离株ＹＧ９７经１０日龄鸡胚增殖后纯化，提取病毒的基因组ＲＮＡ，采用ＲＴ－ＰＣＲ一次性扩增出与预期设计的１．７ｋｂ大小相符的特异性条带。将扩增产物提纯后克隆入ｐＣＥＭ＾Ｒ载体，经转化、筛选及酶切鉴定后，初步获得了含鹅副粘病毒Ｆ基因的阳性克隆。将所获得的阳性重组质粒进一步进行了序列鉴定。序列分析表明扩增的Ｆ基因片段的长度为１６９５ｂｐ，共编码５３３个氨基酸，Ｆ蛋白裂解位点的氨基酸顺序为＾１１２Ｒ－Ｒ－Ｑ－Ｋ－Ｐ－Ｆ＾１１７，与ＮＤＶ强毒株特征相符，同时也与鹅副粘病毒分离株致病性实验结果相符，同源性分析表明，与国内标准强毒株Ｆ４８Ｅ９的核苷酸同源性为８６％，与国内外其它毒株的核苷酸同源性在８２％～８９％之间，表明该毒株相对于经典的ＮＤＶ在Ｆ片段上发生了较大的变异。

Cloning and Sequencing of Fusion Glycoprotein Gene of Goose's Paramyxovirus

The Goose's Paramyxovirus isolate YG97 was propagated in 10_day_old chicken embryos.The allantonic fluids of the virus were purified and the genomic RNA was extracted.The F gene of the virus has been successfully amplified by RT_PCR,and further cloned into pGEM R_T vector,and a positive recombinant plasmid was obtained by restriction enzyme analysis.The sequence analysis showed that the nucleotide sequence of this F gene was 1695 bp and encoding a protein of 553 amino acids.The amino acid sequence of cleavage site region was 112 R_R_Q_K_R_F 117 ,matching to virulent NDV strains,and at the same time it was consistent with the pathogenicity test of the isolates.Sequence comparison with the published Chinese standard virulent strains F48E9,the homology of the nucleotide was 86%,and the homology compaired with other NDV strains was between 82% and 89%.The results indicated the mutation of the F gene between the Goose's Paramyxovirus and the classic NDV varied to a great extent.