我国SARS病毒BJ01株基因组亚cDNA克隆的构建与鉴定

Construction and identification of genomic cDNA subclones of SARS-CoV BJ01 strain in China

目的 :构建我国SARS病毒BJ0 1株基因组全序列的亚cDNA克隆 ,为进一步构建该毒株的全长感染性cDNA克隆奠定基础。方法 :根据BJ0 1株病毒基因组序列中含有的特异酶切位点 ,利用DNAstar软件设计 7对引物 ,然后通过RT PCR扩增出覆盖病毒基因组全长的 7个cDNA片段 ,并分别将其克隆至pGEM TEasy或Topo载体。结果与结论 :已构建出SARS CoVBJ0 1株基因组的 7个亚cDNA克隆 ,经酶切鉴定和序列测定表明 ,所获得的cDNA克隆为BJ0 1株病毒的特异序列。

Objective: To get the cDNA subclones spanning the entire genome of SARS coronavirus BJ01 strain, and lay the foundation for construction of genomic full-length infectious cDNA clone. Methods: According to the restriction endonuclease sites in viral genome of BJ01 strain, seven primer pairs were designed by DNAstar software and the cDNA fragments were amplified by RT-PCR.The amplified products were purified and then cloned into pGEM-T Easy or Topo vectors. Results and Conclusions: Seven cDNA subclones covering the viral genome were obtained. The subclones were sequenced and proved to be consistent with SARS-CoV BJ01 strain.