利用反向遗传操作技术拯救重组新城疫病毒

Rescue of recombinant Newcastle disease virus by reverse genetic system

将新城疫病毒(Newcastle disease virus,NDV)Anhinga株的全长cDNA的克隆质粒以及L、P、NP的表达质粒共转染稳定表达T7 RNA聚合酶的BSRT7/5细胞,得到拯救病毒。通过RT-PCR扩增、酶切鉴定、测序验证拯救病毒中分子标签的存在,并通过血凝试验测定病毒的血凝效价、测定蚀斑形成单位确定病毒滴度,从而证明病毒拯救成功。新城疫病毒Anhinga株的拯救成功为该病毒进行结构基因研究和疫苗研制奠定了基础。

The plasmid of the full-length Newcastle disease virus （NDV） cDNA from Anhinga strain was cotransfected with helper plasmids encoding viral nucleoprotein, phosphoprotein and large polymerase protein into BSR T7/5 cells stably expressing T7 RNA polymerase. The virus was rescued and amplified by inoculation of the supernatant from the transfected cells into the allantoic cavity of specific-pathogen free chicken embryos. By RT-PCR amplification, restriction enzyme digestion and sequencing confirmation, it identified the unique molecular tag of the rescued virus. Virus HA titer was also measured through the hemagglutination test （HA test）, and determined viral titers by plaque-forming units. Thus, it concluded that the virus was rescued successfully from cDNA. The successful rescue of NDV Anhinga strain established a useful platform for the further study of NDV and development of recombinant vaccines.