猪生殖和呼吸综合征病毒B_(13)株ORF7基因的克隆及在杆状病毒系统中的表达

Cloning, Sequencing and Expression of ORF7 Gene of Porcine Reproductive and Respiratory Syndrome Virus B_(13) Strain in a Baculovirus Expression System

利用单管RT PCR方法扩增猪生殖和呼吸综合征病毒 (PRRSV)分离株B13 株包括ORF7基因的片段 ,并对其序列进行了测定 ,结果PRRSV分离株B13 ORF7基因长度为 384bp ,编码 12 8个氨基酸组成的 15kD蛋白。与已发表的PRRSVLV株、VR 2 332株进行同源性比较 ,发现核苷酸同源性分别为 99 2 %、5 9 4% ;氨基酸同源性分别为98 4%、5 4 7%。表明PRRSV分离株B13 在基因结构上可能与LV株同属于欧洲亚群。同时构建了重组转移载体质粒 pAcGHLT B ORF7,用该重组转移载体质粒与线性化苜蓿丫纹夜蛾核型多角体病毒 (AcMNPV SVI-G)基因组DNA(baculogoldlinearizedbaculovirusDNA)共转染草地夜蛾 (Spodoptcrafrugiperda ,Sf9)细胞 ,得到重组病毒AcMNPV OCC- GST 6xHis ORF7。在感染了重组病毒的Sf9细胞中检测到分子量为 46kD的ORF7基因的GST融合蛋白表达产物 ,能被猪抗PRRSVB13 株多克隆血清所特异识别。

The DNA fragments were amplified separately by one-tube RT-PCR, in which contained the complete ORF 7 gene of B 13 strain of PRRSV. The ORF 7 gene of B 13 strain of PRRSV was sequenced by automatic sequencing. The sequence of ORF 7 gene consisted of 384bp. The predicted polypeptide of ORF 7 gene consisted of 128 amino acids, and its molecular weight was about 15kD. After comparing the ORF 7 gene of B 13 strain with that of LV and VR-2332 which have been published previously, it was found that the nucleotide sequence of B 13 was 99.2% and 59.4% identical to LV and VR-2332 respectively, the predicted amino acid sequence was 98.4% and 54.7% identical with LV and VR-2332 respectively. The results showed that the B 13 strain of PRSV and the LV strain both may belong to a same subgroup, the European subgroup. Meanwhile, recombinant plasmid vector pAcGHLT-B-ORF 7 was constructed. After cotransfecting the cultured Sf9 cells with pAcGHLT-B-ORF 7 and the linearized genome DNA of AcNPV-SVI-G and TnNPV-SVI-G respectively, the recombinant virus AcNPV-OCC--GST-6×His-ORF 7 was obtained. In the Sf9 cells infected with the recombinant virus, the expressed ORF 7-GST fusion protein of B 13 strain with molecular weight about 46kD was successfully detected by Western-blot using anti-PRRSV B 13 polyclonal sera. The results of this study lay a foundation for developing new PRRS diagnostic antigen.