PCV-2 ORF2和PRRSV ORF5基因的扩增与双基因真核表达载体的构建

Sequence Analysis of ORF2 Gene of PCV-2 Strain SD1 and ORF5 Gene of PRRSV Strain SD2 and Construction of Eukaryotic Expressing Vector of ORF2 and ORF5

根据GenBank中猪圆环病毒2型ORF2基因序列和猪繁殖与呼吸综合征病毒ORF5基因序列,分别设计一对引物,应用PCR和RT-PCR扩增出PCV-2 ORF2基因和PRRSV ORF5基因。将PCR产物ORF2经NheⅠ/EcoRⅠ双酶切回收后插入真核表达载体pIRES得到pIRES-ORF2,RT-PCR产物ORF5经NotⅠ/XbaⅠ双酶切回收后插入pIRES-ORF2构建重组质粒pIRES-ORF2/ORF5。对此质粒中的插入片段,进行PCR RT-PCR及双酶切鉴定,同时对插入序列进行测序分析,表明SD1株ORF2与国内多种毒株的同源性为99%,pIRES-ORF2ORF5转移载体ORF5基因的核苷酸推导氨基酸与北美洲原型(VR-2332株)氨基酸同源性为99%。此重组表达载体的成功构建,为进一步研究PCV-2 ORF2及PRRSV ORF5编码蛋白的生物学活性及研究猪圆环病毒和猪繁殖与呼吸综合征病毒二联核酸疫苗奠定了基础。

According to the published ORF2 gene sequence of PCV-2,two primers were designed, and ORF2 gene was amplified from suspected PMWS sample using PCR. With the same way,ORF5 gene sequence of PRRSV was amplified using RT-PCR. Then first , the PCR product ORF2 was digested with Nhe Ⅰ / Eco RⅠ ,ORF2 gene was purified, and introduced into eukaryotic expressing vector PIRES between Nhe Ⅰ and EcoR Ⅰ enzyme site. Secend,the RT-PCR product ORF5 was introduced into PIRES by the same way between Not Ⅰ and Xba Ⅰ. Then, pIRES ORF2/ORF5 recombinant plasmid was constructed. Finally, we succeeded in constructing pIRES ORF2/ORF5 identified by PCR; double digestion and nucleotide sequencing. then ORF2 and ORF5 in pIRES ORF2/ORF5 were sequenced and compared with other PCV-2 and PRRSV isolates in GenBank separately. The results demonstrated that the ORF2 gene was closely related with many isolates,and the ORF5 gene was closely related with a USA isolate VR-2332, the amino acid sequence were 99 %. Further research is underway to study the biological activity of ORF2 and ORF5 protein and this will be used for future gene vaccine.