鸭源新城疫F基因和鸭圆环病毒Cap基因融合表达DNA疫苗的构建

Construction of Recombinant Plasmid Coexpressing Cap Gene of Duck Circovirus and F Gene of NDV Isolated from Duck

设计了2对特异性引物,PCR扩增出鸭源NDV分离株GX2011/19的F基因和DuCV的Cap基因,并将其克隆到真核表达载体pcDNA3.1,成功构建重组融合表达质粒pcDNA3.1-F-Cap。经酶切和测序鉴定正确后,将重组质粒pcDNA3.1-F-Cap转染Vero细胞,采用间接免疫荧光检测到目的基因的体外表达。将pcDNA3.1-F-Cap作为DNA疫苗对2周龄的SPF雏鸭以胸部肌肉多点注射的方式进行免疫。分3个实验组,每组接种剂量分别为100、200和300μg/只,另设2组为载体质粒空白组和灭活疫苗组。ELISA检测抗体水平,试验结果显示:各DNA疫苗免疫组的NDV抗体和DuCV抗体水平明显高于载体空白对照组,差异显著(P<0.05);二免后不同DNA剂量组间的抗体水平随着免疫剂量的增大而增加,差异显著(P<0.05);于第二次免疫后2周,使用分离株GX2011/19以滴鼻的方式进行攻毒试验。接种pcDNA3.1-F-Cap剂量为300μg/只免疫组的保护率为40%,高于200μg/只组(30%)和100μg/只组(10%)。研究结果表明,构建的DNA疫苗对新城疫病毒的强毒攻击具有一定的抵抗力,此疫苗的保护率随着免疫剂量的增大而有所增加。

Two pairs of specific primers were designed,and then the amplified F and Cap genes were iaserted into pcDNA3.1 to construct the recombined plasmid pcDNA3.1-F-Cap.The recombined plasmid was confirmed by enzyme digestion and sequencing.And subsequently the recombined plasmid was transfected into Vero cells.The in vitro expression of the target gene was identified by the immunofluorescence test.The recombined plasmid pcDNA3.1-F-Cap as DNA vaccines was injected into two-week-old SPF duck.These ducks were divided into five groups with different doses,ranging from 300 μg/duck to 100 μg/duck（300,200 and 100 μg/duck）.Besides,another two groups were injected with pcDNA3.1as negative control and GX19/2011strain oli-emulsion as positive control respectively.Antibody levels were tested by ELISA method,and the results showed that antibody levels of NDV and DuCV of each immunized group injected with DNA vaccines pcD-NA3.1-F-Cap were both significantly higher than those of the negative contrl （P ＜0.05）.The antibody levels of immunization groups increased with the increase of immune dose （P ＜0.05）.Two weeks after the second immunization,challenge experiment was done.The results of protective immunity showed that DNA vaccines pcDNA3.1-F-Cap group immunized 300 μg/duck exhibited 40％ of protective rate,higher than that of 200 μg（30％） and 100 μg（10％）.The results showed that the DNA vaccine had certain resistance to virulent NDV and the protection rate of this vaccine increased with dose.