C3d对减毒沙门氏菌介导的NDV DNA疫苗诱导的特异性体液免疫应答的增强作用

采用PCR技术从重组质粒pVAX1-F扩增出新城疫病毒F48E8株的融合蛋白(F)基因，将其克隆入含有3 拷贝C3d 编码基因的pTR-C3d3质粒中，获得重组质粒pTR-F-C3d3。将F-C3d3基因片段从pTR-F-C3d3中切出，克隆入真核表达质粒pVAX1中，获得重组真核表达质粒pVAX1-F-C3d3。将pVAX1-F-C3d3转化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(pVAX1-F-C3d3)。重组菌分别以1×109 CFU的剂量两次口服免疫1日龄SPF鸡，结果显示，SL7207(pVAX1-F-C3d3)激发产生的血清抗体效价与SL7207 (pVAX1-F)免疫组之间存在显著性差异(P < 0.05)。SL7207(pVAX1-F-C3d3)免疫组的小肠黏膜抗体效价高于SL7207(pVAX1-F)免疫组，但两者间差异不显著(P > 0.05)。提示C3d可增强减毒沙门氏菌介导的NDV DNA疫苗诱导的特异性体液免疫应答水平，这为提高基于减毒沙门氏菌的NDV基因工程疫苗的免疫效果提供了新的途径。

Enhancement of the Humoral Immune Response against the Attenuated Salmonella typhimurium Harbouring Newcastle disease virus DNA Vaccine by C3d Molecule

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant eukaryotic expression plasmid pVAX1-F, and subcloned into the plasmid pTR-C3d3 containing three copies of C3d. The F-C3d3 gene was identified by sequencing and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX1-F-C3d3 was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207(pVAX1-F-C3d3). One-day-old SPF chickens were immunized orally with SL7207(pVAX1-F-C3d3) at the dosage of 1×109 CFU and boosted two weeks later with the same dose. The serum antibodies responses specific to NDV were observed in chickens immunized with SL7207(pVAX1-F-C3d3) on day 14 and 28, and their levels were significantly higher than that of SL7207(pVAX1-F) (P < 0.05). Intestinal mucosal immune response were observed in chickens immunized with recombinant bacteria on day 28, and their levels were significantly higher than that of negative and positive control (P < 0.05), but there was not significance between the groups SL7207(pVAX1-F-C3d3) and SL7207(pVAX1-F). These results indicated that three copies C3d could enhance humoral immune response against NDV induced by attenuated Salmonella typhimurium harbouring DNA vaccine. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.