减蛋下降综合征病毒重组 Knob-S 基因在杆状病毒系统中的表达及其免疫原性鉴定

摘要

为以杆状病毒系统在Sf9细胞中表达含减蛋下降综合征病毒( EDSV)重组Knob-S蛋白质来制备亚单位疫苗,采用PCR方法从鸭胚繁殖的EDSV尿囊液中扩增Knob-S基因,并克隆到pFastBac1中,经转化DH10Bac获得重组rBac-Knob-S。将rBac-Knob-S转染Sf9细胞获得重组杆状病毒,利用重组杆状病毒感染Sf9细胞表达重组蛋白质。重组Knob-S蛋白质经Western blotting和间接免疫荧光试验鉴定均为阳性。将重组Knob-S蛋白质制备成疫苗,按1羽0.3 ml(含50μg的重组病毒)免疫6周龄非免海兰褐雏鸡,在免疫后第2周可检测到血凝抑制抗体效价达8.5 lg2,在第3周达到高峰(9.5 lg2),表明重组Knob-S蛋白质具有较好的免疫原性。%To develop egg drop syndrome virus subunit vaccine ( EDSV) based on its recombinant Knob-S proteins expressed in Sf9 cell, the Knob-S gene of EDSV was amplified from allantoic fluids of duck embryos by PCR, and cloned into pFastBac-1 before transformation into DH10Bac to construct rBac-Knob-S bacmid. The recombinant Knob-S proteins were expressed in Sf9 cell by infection of the recombinant baculovirus derived from the transfection of the bacmids in Sf9 cell. The recombinant Knob-S proteins were identified by Western blotting and indirect immunofluorescence assay. Groups of 6-week-old nonimmuned HY-Line brown chickens were inoculated with vaccines containing the recombinant Knob-S proteins. The serum antibody levels reached 8. 5 lg2 2 week post-vaccination, and rose to the peak of 9. 5 lg2 3 week post-vaccination determined by hemagglutinin inhibi-tion test, indicating that recombinant Knob-S proteins had good immunogenicity for chickens.