犬瘟热病毒融合蛋白、血凝蛋白、基质蛋白和核衣壳蛋白基因DNA联合免疫的研究

Immunogenicity of the combinations of recombinant plasmids encoding fusion, hemagglutinin, matrix and nucleocapsid genes of canine distemper virus in dogs

为研究犬瘟热病毒(CDV)融合蛋白(F)、血凝蛋白(H)、基质膜蛋白(M)和核衣壳蛋白(N)基因核酸联合免疫的效力,本研究分别构建了表达经哺乳动物密码子优化的CDV F、H、M和N蛋白基因的真核重组表达质粒p CAGG-CDVF、p CAGG-CDVH、p CAGG-CDVM和p CAGG-CDVN;将其分别转染BHK-21细胞后通过间接免疫荧光试验表明,目的蛋白均获得正确表达。此外,将等量混合的重组质粒p CAGG-CDVF、p CAGG-CDVH、p CAGG-CDVM(3组份DNA疫苗)或重组质粒p CAGG-CDVF、p CAGG-CDVH、p CAGG-CDVM、p CAGG-CDVN(4组份DNA疫苗),分别以500μg/只经肌肉注射途径免疫A组(6只)或B组(6只)比格犬,间隔4周以相同剂量、途径加强免疫一次,并于初次免疫前、后不同时间检测血清CDV中和抗体。中和试验结果显示,A组和B组免疫犬,二免4周时,CDV中和抗体滴度平均值达到峰值,分别为6 log2和5.64 log2;在初免54周时,CDV中和抗体滴度均仍然维持5 log2。因此,3组份DNA疫苗为具有良好应用前景的犬瘟热候选疫苗。

To evaluate the immunogenicity of DNA vaccines expressing Fusion（F）, Hemagglutinin（H）, Matrix（M） and Nucleocapsid（N） proteins of canine distemper virus（CDV） in dogs, the F, H, M and N genes of CDV were synthesized with coding optimized according to mammalian favored codons and cloned into eukaryotic expression vector p CAGGS to construct recombinant plasmid p CAGG-CDVF, p CAGG-CDVH, p CAGG-CDVM and p CAGG-CDVN, and the transient expressions of F, H,M and N proteins were verified by transfecting the recombinant plasmids into BHK-21 cells, respectively, and detected by indirect immunofluorescence assay. In addition, 500 μg of p CAGG-CDVF, p CAGG-CDVH and p CAGG-CDVM（3-gene combined DNA vaccine） and 500 μg of p CAGG-CDVF, p CAGG-CDVH, p CAGG-CDVM and p CAGG-CDVN（4-gene combined DNA vaccine）were immunized to beagle dogs in group A and group B, respectively, via intramuscular inoculation for primary and booster vaccination at 4 weeks interval. The results of virus neutralization demonstrated that the average of neutralization antibody（NA）titers in dogs both in group A and group B reached the peak level of 6 log2 and 5.64 log2 at 4 weeks post booster immunization,which remained 5 log2 for 54 weeks after primer immunization. Therefore, the combinations of the 3-gene combined DNA vaccine have the premising to be served as a DNA vaccine against CDV infection in dogs.