猪霍乱沙门菌C500运载CSFV新型基因疫苗的稳定性及免疫安全性

为研究猪霍乱沙门菌C500(S.C500)运载猪瘟病毒(CSFV)新型基因疫苗的稳定性与免疫安全性,将表达载体pCB-ME2-IL-15、pCC-ME2-IL-15、pCI-ME2-IL-15及pCI-neo转入S.C500,采用体外增殖试验考察不同表达载体在S.C500中的稳定性;采用携质粒S.C500对ST细胞的粘附、侵袭力和增殖试验研究不同表达载体对运载体生物学特性的影响;采用BALB/c小鼠口服免疫试验研究表达质粒在体内的稳定性和运载体对小鼠的安全性。结果显示:体外增殖中pCB-ME2-IL-15、pCC-ME2-IL-15和pCI-ME2-IL-15稳定性好于pCI-neo,说明外源目的基因正调表达质粒在运载体内的稳定性;空运载体S.C500对ST细胞的粘附率和侵袭率均高于4组含不同质粒的菌株,而4组含不同质粒的菌株之间亦有差异;空运载菌在ST细胞内的增殖能力与4组含不同质粒的菌株之间亦有显著性差异;分别对携带表达载体pCB-ME2-IL-15、pCC-ME2-IL-15和pCI-ME2-IL-15菌株以每只2×108CFU/0.2 mL的剂量进行小鼠口服免疫,腹泻率依次为0%、8.33%和25%。在第7和14天,使用DHL/Amp琼脂培养平板,均可从肝脏、脾脏、十二指肠和新鲜粪便样品中分离到重组菌。试验提示采用猪霍乱沙门菌C500运载CSFV新型基因疫苗免疫BALB/c小鼠具有相对稳定性和免疫安全性。

Stability and immunization safety of the new gene vaccine of CSFV using Salmonella choleraesuis C500 strain as transporter

The study was performed to investigate the stability and immunization safety of the new gene vaccine of classical swine fever virus(CSFV) using Salmonella choleraesuis C500(S.C500) strain as transporter.These expression vectors pCB-ME2-IL-15,pCC-ME2-IL-15,pCI-ME2-IL-15 and pCI-neo were transfered into S.C500 strain.The stabilities of different expression vectors in S.C500 were analyzed by proliferation in vitro.To confirm whether the biological characteristics of recombinant strains were affected by these expression vectors or not,the investigation on the effects of S.C500 carrying these plasmids on adhesiveness,invasiveness and proliferation to ST cells was conducted.At the same time,the oral immunization in BALB/c mice was carried out to investigate the stability in vivo of expression vectors and the security of these recombinant strains to mice.The results indicated that the stabilities of pCB-ME2-IL-15,pCC-ME2-IL-15 and pCI-ME2-IL-15 were better than that of the vector pCI-neo,which suggested that the exogenous gene would enhance the stability of the expression plasmids in the transporter.The ratioes of adhesiveness and invasiveness of S.C500 to ST cells were higher than those of other four recombinant strains,and there existed a difference among the four strains.As to the proliferation abilities in ST cells,there were also distinct differences between these four strains and S.C500.These recombinant strains carrying expression vectors pCB-ME2-IL-15,pCC-ME2-IL-15 and pCI-ME2-IL-15 were orally immunized to BALB/c mice with the dose of 2×108 CFU/0.2 mL.The diarrhea rates were 0%,8.33% and 25%,respectively.At the third and seventh days after oral immunization,all these recombinant strains could be isolated from liver,spleen,duodenum and fresh fecal samples of mice by DHL/Amp agar plate method.The results demonstrated that the immunity with S.choleraesuis C500 carrying the new gene vaccine of CSFV was relatively stable and safe.