摘要
通过RT-PCR方法检测到一份猪繁殖与呼吸综合征病毒(PRRSV)阳性肺组织病料。将该病料研磨、过滤后感染Marc-145细胞,连续传代都能产生明显的细胞病变。为鉴定细胞病变是否由PRRSV引起,抽提细胞总RNA,通过RT-PCR的方法成功的检测到了PRRSV基因。同时用间接免疫荧光方法证实了感染的细胞内有PRRSV N蛋白的表达。将成功分离的毒株命名为GXYL1403株。对分离株的ORF5基因和Nsp2高变区进行扩增、克隆和遗传进化分析,结果发现该毒株Nsp2高变区与NCBI登录的参考序列VR-2332氨基酸同源性为68.6%,JXA1的同源性为90.8%。通过ORF5遗传进化分析该分毒株为北美型PRRSV,且与高致病性PRRSV分布在同一个小分支上。Nsp2序列对比显示该分离株Nsp2蛋白含有高致病性PRRSV特异的1+29个氨基酸的缺失。特别的是,在缺失29个氨基酸区域的上游,含有连续20个氨基酸缺失,在下游更出现了连续73个氨基酸的缺失。该PRRSV毒株的分离和Nsp2蛋白部分序列的缺失鉴定,为下一步研究Nsp2部分氨基酸缺失对病毒生物学特性影响奠定了基础。
A lung tissue sample was detected with RT-PCR for porcine reproductive and respiratory syn-drome virus.To isolate the PRRSV from the lung tissue,tissue sample that was crumbled,filtrated and in-oculated into Marc-145 cells and subcultured serially for three times.PRRSV typical CPE was observed on Marc-145 cells at all three passages.PRRSV RNA of supernatant collected from infected Marc-145 cells was detected by RT-PCR.PRRSV N protein in Marc-145 cells inoculated with tissue sample was detected by indirect immunofluorescence assay（IFA）confirming the CPE was producted by PRRSV.The PRRSV strain was named PRRSV GXYL1403.The ORF5 and hypervariable region of Nsp2 protein were amplified, cloned and sequenced.Phylogenetic analysis of ORF5 of PRRSV showed that GXYL1403 strain belonged to North American genotype.Sequence analysis of hypervariable region of Nsp2 gene showed that GXYL1403 strain had 68.6% amino acid identity with VR-2332 strain and 90.8% with JXA1.More importantly,GX-YL1403 strain contained a deletion of 20 aa in Nsp2 coding region,which was located in upstream of 29 aa deletion.Beside,a continuous deletion of 73aa in Nsp2 region emerged following the 29 aa deletion.Charac-terization of PRRSV GXYL1403 strain was useful to study the effect of partial aa deletion of Nsp2 on its biological activities.
出处
《动物医学进展》
北大核心
2015年第10期34-38,共5页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31372444)
广西大学大学生实验技能和科技创新能力训练基金项目(SYJN20131705)
广西大学科研基金项目(XGZ130959)
关键词
高致病性猪繁殖与呼吸综合征病毒
分离和鉴定
NSP2基因
氨基酸缺失
highlyand identification
pathogenic porcine reproductive and respiratory syndrome virus（HP-PRRSV） ~isolationNsp2 gene
amino acid deletion
