犬瘟热病毒强毒株在Vero-SLAM细胞上传代后的基因突变分析

Genetic mutation analysis of complete genome of canine distemper virus virulent strain passaged on Vero-SLAM cells

为了了解犬瘟热病毒强毒株全基因组序列在Vero-SLAM细胞上传代培养后的突变情况,本试验利用犬瘟热病毒强毒分离株HeBei株第12代细胞毒,通过RT-PCR方法分段扩增犬瘟热病毒全基因,对扩增出的各个片段进行克隆和鉴定,并应用SeqMan程序将所测得的序列进行拼接成Hebei株全长cDNA序列。结果表明,全基因与原始序列相比有24处碱基突变,总突变率为0.15%,其中3处为非编号码区的突变,其余21处在编码区,碱基突变率最高的蛋白基因为F(0.36%),最低为N(无突变)。氨基酸突变率分别为N蛋白0.00%;P蛋白0.39%;M蛋白0.30%;F蛋白0.60%;H蛋白0.50%;L蛋白0.18%。表明犬瘟热病毒强毒株经Vero-SLAM细胞传代培养后基因突变主要发生在F蛋白基因上。

In order to understand the genetic mutation of virulent canine distemper virus cultured on Vero-SLAM cells,Hebei strain,the virulent isolate of canine distemper virus,was cultured on Vero-SLAM cells for 12 passages and subjected for gene amplification by RT-PCR method. Each amplified fragment was cloned and identified,and the full-length cDNA sequence of Hebei strain was spliced using Seq Man program. The results showed that 24 nucleotide mutations were found comparing with the original sequence,among of which,3 mutated nucleotides were located at non-coding regions,and the remaining 21 nucleotides were located at the encoding regions. F protein gene showed the highest nucleotide mutation rate（ 0. 36%）,while N protein gene showed no nucleotide mutation. The mutation rate of amino acid of N,P,M,F,H,L protein is 0. 00%,0. 39%,0. 30%,0. 60%,0. 50% and 0.18%,respectively. It indicates that minor genetic mutation appears during the passage of canine distemper virus virulent strain on Vero-SLAM cells,and mutations occur mainly on the F protein gene.