稳定携带新城疫病毒DNA疫苗减毒沙门氏菌的构建及其免疫原性

Construction and Immunogenicity of Attenuated Salmonella Typhimurium Stably Harbouring DNA Vaccine against Newcastle Disease Virus

采用PCR技术从重组质粒pVAX1-F中扩增出新城疫病毒JS5株的融合蛋白(F)基因,将其克隆入真核表达质粒pmcDNA3.1+中,获得重组表达质粒pmcDNA3.1-F。通过电穿孔转化法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pmcDNA3.1-F)。体内、体外试验结果表明,重组质粒pmcDNA3.1-F在沙门氏菌中的稳定性显著高于pcDNA3.1-F。将重组菌SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)分别以1×109CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对新城疫病毒F蛋白的血清抗体和黏膜抗体。重组菌以5×109CFU剂量两次口服免疫4日龄SPF鸡,免疫鸡产生的针对新城疫病毒F蛋白的血清抗体和小肠黏膜抗体效价水平与空载体组之间存在显著性差异(P<0.05)。免疫保护试验结果显示,SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)免疫组的免疫保护率均与空载体组之间存在显著性差异(P<0.05),且SL7207(pmcDNA3.1-F)免疫组的保护率较SL7207(pcDNA3.1-F)免疫组提高了20.0%,说明稳定携带新城疫病毒DNA疫苗的减毒沙门氏菌具有良好的免疫原性和免疫保护性。

The fusion protein （F） gene of Newcastle disease virus was amplified by polymerase chain reaction （PCR） from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3.1＋. The F gene was identified by sequencing. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207（pmcDNA3. I-F）. In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3.1-F was apparently higher than that of pcDNA3.1-F in SL7207. In order to compare the immune response induced by these two recombinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2×10^9 CFU respectively. Both SL7207（pcDNA3. I-F） and SL7207（pmcDNA3. I-F） initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207（pcDNA3. I-F） and SL7207（pmcDNA3. I-F） at the dosage of 5×10^9 CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207（pmcDNA3. I-F） had the protective rate of 70.0%, higher than that of the SL7207 （pcDNA3. I-F） with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.