麻鸡新城疫病毒SX-1株F和HN基因共表达蛋白的免疫原性

Immune experiment of co-expresstion F and HN protein of partridge chickens Newcastle disease virus strain SX-1

用PCR扩增麻鸡新城疫病毒SX-1株F基因和HN基因,并以由15个氨基酸构成的短肽为Linker将F及HN基因片段体外连接,与转座载体PFastBacⅠ连接,获得重组质粒PFastBacF-HN,并转化到DH10Bac宿主菌,将目的基因定向插入到Bacmid质粒中,经筛选获得的重组Bacmid F-HN质粒转染Sf-9昆虫细胞,进行表达检测,表达产物间接免疫荧光试验阳性,经SDS-PAGE、Western-blot检测显示,获得相对分子质量约123 000的蛋白特异条带,说明F-HN重组蛋白表达成功。将共表达蛋白进行进行动物试验,间接血凝试验表明雏鸡免疫14d后在体内可检测到特异性抗体,共表达蛋白二次免疫雏鸡后,对NDV强毒的攻毒保护率为80%,这一结果提示利用F-HN基因构建的亚单位疫苗具有重要的开发应用价值。

F gene and HN gene of partridge chickens Newcastle disease virus strain SX-1 were amplified and connected by a flexible short peptide Linker in vitro. The constructed plasmid PFastBac F-HN was transformed into the host cell DH10Bac to reconstruct the recombinant plasmid Bacmid F-HN. Then the Bacmid F-HN was transfected into the Sf-9 cells for expression of the F-HN gene. The results of SDS-PAGE,Western-blot and IFA showed that the F-HN co-expression protein was expressed well. Specific antibody were displayed at 14 d after being immuned with the co- expression F-HN protein,and the protective rate of the chicken challenged by NDV standard virulent infection was 80%. The result indicated that the co-expression F-HN protein could be useful for controlling the current epidemic of ND in partridge chickens of China.