携带新城疫病毒DNA疫苗的鼠伤寒沙门氏菌及其安全性与免疫效力  

Safety and efficacy of attenuated Salmonella typhimurium harbouring DNA vaccine against Newcastle disease virus

作　　者：潘志明[1] 焦新安[1] 黄金林[1] 殷月兰[1] 唐丽华[1] 张辉[1] 张晓明[1] 张小荣[2] 刘秀梵[2] 

机构地区：[1]扬州大学生物科学与技术学院 [2]扬州大学农业部畜禽传染病学重点开放实验室,扬州225009

出　　处：《微生物学报》2005年第6期937-941,共5页Acta Microbiologica Sinica

基　　金：国家"863计划"(2002AA245051);国家自然科学基金(30425031;30170700);江苏省自然科学基金(BK2003043)~~

摘　　要：根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计一对引物,通过RTPCR扩增出鹅源新城疫病毒分离株JS5F基因,测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1F。将pVAX1F转化减毒鼠伤寒沙门氏菌SL7207,构建成携带DNA疫苗的重组沙门氏菌SL7207(pVAX1F)。重组菌以不同剂量口服免疫1日龄雏鸡,结果表明,细菌对雏鸡具有良好的安全性,且不影响鸡的增重。将SL7207(pVAX1F)分别以108CFU和109CFU的剂量3次口服免疫1日龄商品代伊莎褐蛋鸡,抗体检测结果显示,在三免后1周,SL7207(pVAX1F)109CFU剂量组的血清抗体效价与空载体组之间存在显著性差异(p<0.05)。重组菌两个剂量组在首免后2周开始出现粘膜抗体,并于二免后2周和3周达到较高水平。免疫保护试验结果显示,SL7207(pVAX1F)109CFU剂量组的保护率为77.27%,与空载体组之间存在显著性差异(p<0.05)。A pair of primers were designed and synthesized according to the previously published sequence of fusion protein （F） gene of Newcastle disease virus（NDV） and used to amplify F gene by reverse-transcription polymerase chain reaction （RT-PCR） from the genomic RNA of a NDV strain JS5 isolated from goose. The PCR product was identified by sequencing. Then recombinant eukaryotic expression vector pVAX1-F was constructed through inserting F gene into MCS of pVAX1. The recombinant plasmid pVAX1-F was transfected in COS-7 cells, and identified for the transient expression of F gene by indirect immunofluorescent assay. Finally, the recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was screened and designated as SL7207 （pVAX1-F）. It was verified that SL7207 （pVAX1-F） as the oral NDV DNA vaccine was safe for chickens after oral immunization at dosage of 10^10 CFU or below. 1-day-old commercial ISA brown chickens were immunized orally with SL7207 （pVAX1-F） at two different dosages（10^9 CFU and 10^8 CFU） on day 1, 14 and 28. On day 7 after the last immunization, no significant difference was observed in the body weight between these two groups （p 〉 0.05 ）, and also no significant difference between those two groups and negative control group （p 〉 0.05）. Since there were maternal antibodies, high ELISA titers of serum antibodies against NDV were detected in the chickens of all groups on day 14. However, the levels of serum antibodies were decreased in the chickens of all groups on day 28, but the anti-NDV antibody response detected in the sera of chickens immunized with SL7207 （pVAX1-F） at the dosage of 109 CFU were increased and significantly higher than the response induced by immunization with SL7207 （pVAX1） on day 35（p 〈 0.05）. Intestinal mucosal immune response was observed in chickens immunized with SL7207 （pVAX1-F） at the dosage of 109 CFU or l0s CFU. The high ELISA titers of antibodies against NDV i

关 键 词：减毒沙门氏菌 DNA疫苗 新城疫病毒 安全性 免疫效力 

分 类 号：S852.61[农业科学—基础兽医学]