摘要
为构建猪繁殖与呼吸综合征病毒(PRRSV)反向遗传操作系统,将PRRSV全基因组分6段克隆,构建获得6个重组质粒,先由A1和A2片段或B1和B2片段连接构成A片段或B片段,然后D、C、B和A片段依次亚克隆入pOKq载体。在基因组5′端和3′端分别加上CMV启动子序列和BGH终止信号肽,全基因组510位核苷酸突变引入遗传标记位点FseⅠ。测序正确的全基因组质粒命名为pOK-A2BCD。再将pOK-A2BCD质粒转染BHK-21细胞,拯救病毒通过PCR、酶切、测序和间接免疫荧光试验(IFA)鉴定,结果表明拯救病毒成功,为进一步研究PRRSV致病性等相关分子机制奠定了基础。
To establish a reverse genetic system of porcine reproductive and respiratory syndrome virus（PRRSV）,an DNA-launched（plasmid DNA transfection-based）reverse genetics system was developed for PRRSV by introduction of ribozyme elements at both termini of the viral genomic cDNA that were placed under the control of a CMV promoter.The strategy for construction of full-length cDNA clones：Capital letters（A1,A2,B1,B2,C,D）represent 6overlapping fragments amplified from XH-GD genome according to the unique restriction enzyme cleavage sites in viral cDNA.An enzyme cleavage site Not I was added to5′end of A fragment,while 41 nucleotides of polyadenosine tail followed by BGH polyadenylation signal and KpnI were added to 3′end of D fragment by PCR mutagenesis.The Fse I in fragment A1 was created by mutation to be the genetic marker.A CMV promoter with T7 promotor preceded the viral genome.The fragments were inserted into the low-copy vector pOKq in the order of D to A.The completed full-length clone was named pOK-A2 BCD.BHK cells were transfected with pOK-A2 BCD,then the rescued viruses were identified by RT-PCR,restricted enzyme digestion,sequencing and IFA assays.The establishment of the reverse genetics system of PRRSV laid the foundation for further study on the molecular basis of the pathogenicity of PRRSV.
出处
《动物医学进展》
北大核心
2016年第11期1-8,共8页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31272564)
现代农业产业技术体系专项资金项目(CARS-36)
海南省科学事业费项目(13-214002-0002)
