稳定携带H5亚型禽流感病毒候选DNA疫苗减毒沙门氏菌的构建及其免疫原性

采用PCR技术从重组质粒pVAX1-HA扩增出禽流感病毒JSGO(H5N1)株的血凝素(HA)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-HA。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-HA)。经体内体外试验证实,重组质粒pmcDNA3.1-HA在沙门氏菌中的稳定性显著高于pcDNA3.1-HA。将重组菌SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)分别以2×109CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对禽流感病毒HA蛋白的黏膜抗体。重组菌以5×109CFU剂量两次口服免疫试验鸡,免疫鸡的小肠样品中可测到针对禽流感病毒HA蛋白的黏膜抗体,且SL7207*(pmcDNA3.1-HA)免疫组的抗体效价高于SL7207*(pcDNA3.1-HA)免疫组。免疫保护试验结果显示,SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)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-HA)免疫组的保护率较SL7207*(pcDNA3.1-HA)免疫组提高了22.6%,说明稳定携带H5亚型禽流感病毒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。

Construction and immunogenicity of attenuated Salmonella typhimurium harbouring stable DNA vaccine against H5 subtype of Avian influenza virus

The hemagglutinin protein (HA) gene of avian influenza virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-HA, and subcloned into eukaryotic expression vector pmcDNA3.1+. The HA gene was identified by sequencing. The recombinant plasmids were transformed into attenuated Salmonella typhimurium SL7207*, and the recombinants were designated as SL7207(pmcDNA3.1-HA). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3.1-HA is apparently higher than pcDNA3.1-HA in SL7207*. In order to compare the immune response induced by those two recombinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2×10⁹CFU respectively. Both SL7207*(pcDNA3.1-HA) and SL7207*(pmcDNA3.1-HA) initiated HA-specific mucosal antibodies in immunized mice. Furthermore, commercial ISA brown chickens were immunized with SL7207*(pcDNA3.1-HA) and SL7207*(pmcDNA3.1-HA) at the dosage of 5×10⁹CFU and boosted two weeks later with the same dose. Intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive control. The result of protective immunity showed that the chicken immunized with SL7207*(pmcDNA3.1-HA) had the protective rate of 79.3%, higher than that of SL7207*(pcDNA3.1-HA) with 56.7%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of avian influenza in the poultry.