基于DNA-launched的猪繁殖与呼吸综合征病毒反向遗传操作系统的建立

Establishment of DNA-launched Reverse Genetics System of PRRSV

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.