四株新城疫病毒流行株F基因的遗传变异分析

Genetic Variance Analysis of F Gene of 4 Strains Newcastle Disease Virus Isolates

设计并合成了两对寡核苷酸引物 ,P1和P2 用于扩增新城疫病毒 (NDV)全长F基因 ,P3 和P4 用于扩增NDV部分F基因。通过RT_PCR一次性扩增了NDV四平株和长春株的全长F基因和青岛株、昌黎株的部分F基因。将这些基因分别克隆后 ,进行了序列测定和同源率、致病性、疏水性、抗原性和系统发育等比较分析。结果表明 ,四平株和长春株F基因同为176 2bp ,编码 5 5 3个氨基酸。两种F基因推导的氨基酸序列的疏水性相似 ,都具有 3个强疏水区 ,但亲水性有差异 ,抗原表位也有明显的不同 ,说明四平株和长春株F蛋白的抗原性不同。 4株NDVF基因序列 (1～ 813bp)和推导的氨基酸序列 (2 6 1aa)与我国台湾和国外有代表性的NDVF基因相同区域进行了比较和系统发育分析。总体上 ,核苷酸序列同源率在 82 .9%～ 97.5 %之间 ,推导的氨基酸序列同源率在 85 .8%～ 97.7之间 ,长春株与LaSota、TexasGB、Beaudette在同一亚群 ,四平株、昌黎株在同一亚群 ,青岛株为另一亚群。四平株、青岛株和昌黎株F蛋白裂解位点区 (112～ 117aa)的氨基酸序列与强毒株在这一区域的序列相符 ,证明这 3个NDV毒株是强毒株 ,长春株F蛋白裂解位点区的氨基酸序列与弱毒株的序列相符 。

Two pairs of primers were designed and synthetized,in which the P 1 and P 2 were used to amplify the complete F gene of NDV,and the P 3 and P 4 to amplify the part of F gene (1～813bp).The complete F genes of strain Siping,Changchun and the part of F genes of strain Qingdao,Changli were amplified by RT-PCR.These F genes were sequenced after being cloned into plasmids.Sequence analysis showed that the F genes of strain Siping and Changchun were 1762bp long,encoding 553 amino acids.The hydrophobicity of F proteins of the 2 strains was similar,with 3 strong hydrophobic regions,but their antigenicity was different.The 813bp nucleotide sequences and duduced amino acid sequences (261aa) of 4 NDV strains (Siping,Changchun, Qingdao,Changli) were compared respectively with published NDV F genes (10 strains).Their homology was between 82.9%～97.5% in nucleotide sequence,85.8%～97.7% in deduced amino acid sequence.The phylogenetic analysis of nucleotide sequences of F genes (14 NDV strains) showed that strain Changchun and La Sota,Texas GB,Beaudette were in subgroup B,strain Siping and Changli were in subgroup D,strain Qingdao was in a separate subgroup.The amino acid sequence of the cleavage site region (residues 112～117) of the F protein of strain Changchun matched to that of all avirulent NDV strains,the strain was proved to be an avirulent strain.The amino acid sequences of this region of the F proteins of strain Siping,Changli,Qingdao matched to that the of virulent strains,and were confirmed to be virulent strains.