欧洲型PRRSV弱毒株全长基因组cDNA克隆的构建与分析

Construction and analysis of full length genomic cDNA clone for European-type PRRSV low virulent strain

为了获得欧洲型PRRSV弱毒疫苗株AMERVAC-PRRS/A3全基因组序列和病毒基因组全长cDNA克隆,通过RT-PCR技术扩增了AMERVAC-PRRS/A3弱毒株的全基因组,并对基因组cDNA序列进行了测定。根据测序结果,选择合适的酶切位点将各个亚克隆产物逐段克隆入改造过的pBluescriptⅡKS(+)载体中,从而获得了病毒的全长基因组cDNA克隆。测序结果表明,该弱毒株基因组序列全长为15 098 bp(不包括poly(A)尾)。同源性比较结果显示,Ningbo42株ORF7基因(EF473137)、FJ0603株Nsp2基因(EF592535)与该弱毒株的相应基因之间的同源性分别为100%和98%。这些数据暗示,国内欧洲型PRRSV Ningbo42株和FJ0603株的存在与欧洲型PRRSV弱毒疫苗株AMERVAC-PRRS/A3密切相关。测序及酶切鉴定结果表明,欧洲型PRRSV全长基因组cDNA克隆已构建成功。

To obtain genome sequence and full length genomic cDNA clone of the European-type porcine reproductive and respiratory syndrome virus（PRRSV） attenuated vaccine strain AMERVAC-PRRS/ A3,the viral genome was amplified by RT-PCR. The PCR product was cloned into pCR Blunt Ⅱ-TOPO vector to form subclone and then sequenced. The subclone product were cloned into the improved pBluescript Ⅱ KS（＋） vector in proper order by suitable enzyme sites to obtain viral full length genomic cDNA clone. The results showed that the genome was 15 098 bp in length excluding poly（A） tail. The viral ORF7 gene and Nsp2 gene shared 100% and 98% nucleotide identity with ORF7 gene of Ningbo42 strain and Nsp2 gene of FJ0603 strain,respectively. The data suggested that the European-type PRRSV Ningbo42 and FJ0603 strains were closely-related to the European-type PRRSV attenuated vaccine strain AMERVAC- PRRS/A3. Sequencing and enzymatic digestion results showed that the viral full length genomic cDNA clone was constructed successfully.