大熊猫犬冠状病毒部分S基因的克隆与全S基因序列分析

Cloning of partial S gene and sequence analysing of complete S gene of canine coronavirus isolated from giant pandas

从重庆某动物园死亡的大熊猫肝脏中分离到一株病毒,经鉴定为犬冠状病毒(命名为CCVDXMV)。本实验根据CCV K378和CCV Insavc-1株基因序列设计合成了普通PCR引物SF1-SR1和SF2-SR2,以及半套式引物SF3-SR3、SRI3、SFI3、SF4-SR4和SFI4,分段扩增了CCV DXMV株部分S基因,得到了368 bp、792 bp、737 bp、1 178 bp和985 bp 5个基因片段。将纯化的RCR产物分别克隆到pGEM-T载体中,通过筛选和鉴定,获得了5个阳性重组质粒。将重组质粒送生物公司测序,将测得的基因序列与先前测定的该株病毒部分S基因序列,拼接成全S基因序列,并与其它株CCV及TGEV HN2002和FIPV 79-1 146株的S基因序列进行分析比较,绘制进化树。结果表明,该株病毒与CCV K378株的同源性最高,达到99.4%;而与CCV 23/03株的同源性最低,为56.9%;与FIPV和TGEV分别有90.4%和82.1%的同源性。

A strain of virus was isolated and identified to Canine Coronavirus （named from the livers of dead giant pandas from a zoo in Chongqing,China, CCV DXMV）. In this experiment, normal PCR primers SF1-SR1 and SF2-SR2, half nested-PCR primers SF3-SR3, SRI3, SFI3, SF4-SR4 and SFI4 were designed and synthesized according to nucleotide sequences of CCV K378 and Insavc-1 strain. These primers can amplify most partial S gene of CCV DXMV strain. The nucleotide fragments of 368 bp,792 bp,737 bp, 1 178 bp and 985 bp were obtained. The PCR products were purified and then cloned into pGEM-T vectors, and 5 recombinant plasmids were obtained after selected and identified. And recombinant plasmids were sequenced by a biology company. Then the partial S gene of CCV DXMV strain sequenced in the research was connected with the nuleotide sequenced before, and the complete S gene was compared with those of other CCV strains, TGEV HN2002 and FIPV 79-1146. The results showed that the nucleotide sequence homology between CCV DXMV and K378 strain were 99.4 %; and 56.9 %,90.4 % ,82.1% comparing to CCV 23/03, FIPV and TGEV respectively.