猪繁殖与呼吸综合征病毒变异株ORF6基因的克隆与原核表达研究

Cloning and Expression in E.coli of ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus Variant

根据GenBank中美洲型猪繁殖与呼吸综合征病毒(PRRSV)的ORF6基因序列,设计合成一对特异性引物,应用RT-PCR方法扩增出PRRSV的ORF6基因(M基因)。将所扩增片段克隆入原核表达载体pET-32a(+),pET-ORF6重组质粒转化DH5a宿主菌后,经双酶切、PCR鉴定后挑选阳性克隆测序鉴定并对插入的ORF6基因序列进行分析。结果表明,ORF6基因的原核表达载体构建成功。ORF6基因推导的氨基酸与美洲型相应基因的同源性为96.0%～100%,与LV株的同源性为79.9%,系统进化树表明该PRRSV属于美洲型。将构建成功的重组质粒pET-ORF6转化BL21,诱导后经SDS-PAGE和Western-blot分析表明:克隆在HIS标签的膜基质蛋白基因与HIS获得了高效表达,表达的融合蛋白HIS-M分子量约为39kDa,并且有免疫学反应活性。

A pair of primers were designed and synthesized according to the ORF6 gene sequences of American genotype porcine reproductive and respiratory syndrome virus （PRRSV） in GenBank.The ORF6 gene （M gene） of porcine reproductive and respiratory syndrome virus （PRRSV） was amplified by RT-PCR. The amplified gene fIagment was then cloned into the prokaryotic expression vector pET-32a （＋）,the recombinant plasmid was transformed into host cell DH5a ,the selected positive clones were subjected to sequence analysis of the inserted ORF6 gene after double enzyme digestion and PCR identification.The result showed that the construction of the prokaryotic expression vector for ORF6 gene was successful. The deduced amino acids of the cloned ORF6 gene showed 96.0 %-100 % to LV. The phylogenetic trees revealed the strain was clustered within North American genotype. homology to North American strain and 79.9%. The results of SDS-PAGE and Western-blot indicated that the M protein gene cloned in the HIS-Taq was expressed in a high level and the recombinant fusion protein, which was about 39kDa had immunologically reactive activity.