昆虫细胞表达基因ⅦNDVHN基因的免疫效力研究

Expressions of protein HN of Newcastle disease virus in recombinant baculovirus and its antigenicity analysis

利用杆状病毒表达系统对ⅦNDV HN基因进行表达研究。RT-PCR扩增HN基因,将其克隆到p Fast Bac HT A载体中,阳性重组质粒转化DH10Bac感受态细胞,PCR鉴定获得阳性克隆,碱裂解法提取阳性质粒,转染Sf9昆虫细胞,获得含HN基因的重组杆状病毒质粒,重组病毒感染Sf9细胞72 h后,进行SDS-PAGE电泳、间接免疫荧光和Western-blot试验。免疫SPF鸡,间接ELISA测定抗体滴度,攻毒保护试验检测重组HN蛋白保护性。结果显示,目的蛋白HN在昆虫细胞中有特异性表达,该蛋白诱导了高滴度的NDV特异性抗体,具有良好的免疫原性;强毒攻击保护率达到75%,与La sota疫苗组保护率接近,均明显高于阴性对照组。上述结果为NDV新型亚单位疫苗奠定了基础。

The expression of F gene of Newcastle disease virus （NDV） was analyzed using the baculovirus expression system, ttN segments were amplified using RT-PCR and cloned into pFast BacHT A plasmid. The re- combinant plasmid was transformed into DH10Bac competent cells to get the positive recombinant bacmid. The expression of the target gene was detected after 72 h post transfection of Sf9 cell with recombinant bacmid. Serum antibody titer of immunized SPF chickens and the protective properties were determined. The indirect im- munofluorescence assay （IFA） and western-blot test showed that the HN gene expressed in Sf9 cells infected with the recombinant baculovirus. The ELISA results showed that the expressed protein could induce high titer specific antibody against NDV. The protection rate of JSXZ NDV strong virulent strains was 75%, and the protective rate in the La Sota vaccine group was significantly higher than that in the negative control group. These results would lay a foundation for the further study on Newcastle disease virus subunit vaccine.