摘要
为了实现猪繁殖与呼吸综合征病毒(PRRSV)的ORF5和ORF6基因在同一质粒中分别表达各自编码的蛋白,发挥E蛋白的病毒中和优势和M蛋白的细胞免疫优势,将构建成功的pIRES-ORF5/ORF6转移载体用脂质体法转入稳定表达的细胞CHO,经G418加压筛选获得具稳定表达的细胞株。以RT-PCR、SDS-PAGE、Western blot和间接免疫荧光检测目的蛋白的表达情况。结果表明:RT-PCR检测到两种目的基因的转录;SDS-PAGE和West-ern blot检测到同时表达的两种目的蛋白;间接免疫荧光检测到目的蛋白得到表达。
The purpose of this study was to express in CHO the E and M proteins contained in one plasmid vector-pIRES to realize the of neutralizing action of E protein and cell-mediated immunity of M protein. To do this, we designed two pairs of primrs based on the published sequences of pIRES and PRRSV strain SD2 （DQ265739and DQ265740）. Considering the expression level of foreign gene, Kozak sequence was added in the origin codon, pIRES-ORF5/ORF6 was constructed by digestion and ligation and identified by PCR, double digestion and nucleotide sequencing. The recombinant plasmid pIRES-ORF5/ORF6 was transfected into CHO cells using lipofectamine 2000, the stable expressive cells were selected by G418.The ORF5 and ORF6 mRNA in the transfected CHO cells were detected by RT-PCR, the E and M proteins were detected by Western blot and IFA. In conclusion, a recombinant eukaryotic vector involving ORF5 and ORF6 genes was constructed and successfully expressed E and M proteins in CHO cells. This study provides some basic data for further development of PRRS subunit vaccine and will be a new means in the prevention and control of PRRS.
出处
《病毒学报》
CAS
CSCD
北大核心
2006年第6期471-475,共5页
Chinese Journal of Virology
基金
山东省自然基金重大项目(Z2000D03)
