利用DNA Shuffling技术构建重组PRRSV ORF5基因

Construction of recombinant PRRSV ORF5 gene by DNA shuffling technique

摘要:

通过DNA shuffling技术对PRRSV ORF5基因进行改组，研究其对异源毒株交叉中和能力。将获得的嵌合基因△2ORF5与载体pET-32a连接，转化到大肠杆菌Escherichia coli BL21中，经诱导表达获得约42 kDa重组蛋白。将纯化的重组△2ORF5蛋白免疫BALB/c小鼠，制备多克隆抗体，Western blotting试验表明其与4株亲本毒株重组ORF5蛋白具有良好的反应性。病毒感染抑制试验结果表明，多克隆抗体对4株亲本毒株具有较高的抑制率 (>53%)。研究结果为研制新型PRRSV疫苗奠定了基础。

关键词:PRRSV，ORF5基因，DNA shuffling，多克隆抗体，病毒感染抑制试验

Abstract:

Recombinant PRRSV △2ORF5 gene was constructed using DNA shuffling from four genetically different strains of PRRSV to study its heterologous cross-neutralizing ability. The △2ORF5 mutant gene was cloned into the vector pET-32a and transferred into E. coli BL21. SDS-PAGE confirmed that the molecular weight of the recombinant △2ORF5 was about 42?kDa, consistent with the predicted result. Then the purified recombinant protein was injected into BALB/c mouse to obtain polyclonal antibody. Western blotting analysis with mouse-anti-△2ORF5 polyclonal serum indicated that the parental virus recombinant GP5 protein reacted with the specific antibodies. Four parental viruses could be inhibited by the anti-△2ORF5 polyclonal antibody and the inhibition rates were higher than 53%. This work has laid a foundation for further development vaccine for PRRSV.

Key words:PRRSV, ORF5 gene, DNA shuffling, polyclonal antibody, inhibition assay of virus infection