减毒猪霍乱沙门菌ΔcrpΔcyaΔasdC78-1(pYA3493)宿主-载体平衡致死系统的构建及其生物学特性研究  

Construction and characterization of host-vector balanced lethal system of attenuated salmonella choleraesuis ΔcrpΔcyaΔasdC78-1(pYA3493)

作　　者：尚珂[1] 张俊峰[1] 程相朝[1] 张春杰[1] 李银聚[1] 陈桂华[1] 颜云飞 翟崇凯 赵战勤[1] 

机构地区：[1]河南省动物疫病与公共安全院士工作站/河南科技大学动物疫病与公共卫生重点实验室,洛阳471003

出　　处：《中国免疫学杂志》2015年第3期358-363,共6页Chinese Journal of Immunology

基　　金：河南省科技攻关项目(112102110019)

摘　　要：目的:开发应用于猪的稳定携带异源基因的口服活疫苗载体。方法:本研究在减毒猪霍乱沙门菌ΔcrpΔcya C78-1的基础上,运用自杀性质粒pREasd介导的细菌同源重组技术,构建缺失株ΔcrpΔcyaΔasd C78-1,再将携带asd基因的互补质粒p YA3493电转至上述菌株,构建ΔcrpΔcyaΔasd C78-1(p YA3493)宿主-载体平衡致死系统,并进一步研究其生物学特性。结果:PCR及测序结果共同表明ΔcrpΔcyaΔasd C78-1(p YA3493)构建成功。生物学特性检测结果表明ΔcrpΔcyaΔasd C78-1(p YA3493)的血清型与亲本株ΔcrpΔcya C78-1和疫苗株C500相同,并能够稳定遗传缺失型asd基因片段;其生长速度略慢于ΔcrpΔcya C78-1,且二者均明显慢于疫苗株C500;其生化特性结果也与ΔcrpΔcya C78-1基本相同。小鼠口服攻毒试验表明ΔcrpΔcyaΔasd C78-1(p YA3493)的毒力与ΔcrpΔcya C78-1基本相当,但其半数致死量约为疫苗株C500的412倍。结论:上述结果表明,减毒猪霍乱沙门菌ΔcrpΔcyaΔasd C78-1(p YA3493)有潜力作为高效表达外源基因的口服活疫苗载体。Objective： In order to develop an oral live vaccine vector of swine that can stably carry exogenous genes. Methods ： Mutant AcrpAcyaAasdC78-I was constructed by the method of suicide plasmid pREasd-mediated bacteria homologous recombination on the basis of attenuated Salmonella choleraesuis AcrpAcyaC78-1. Complementary plasmid pYA3493 with asd was electrotransformed into the mutant, and then AcrpAcyaAasdC78-1 （ pYA3493 ） host-vector balanced lethal system was constructed. Its biological characteristics were analyzed further. Results： The results of PCR and sequencing showed that AcrpAcyaAasdC78-1 （pYA3493） was constructed suc- cessfully. Biological characteristics showed that the serotype of AcrpAcyaAasdC78-1 （ pYA3493 ） was identical to AcyaAasdC78-1 and vaccine strain C500 and it can stably carry the Aasd gene in vitro;its growth speed was a little slower than AcrpAcyaC78-1 strain, but both of their growth speeds were significantly slower than vaccine strain CS00;the biochemical characteristics of AerpAcyaAasdC78-1 （pYA3493） were basically the same with AcrpAcyaC78-1 strain. Oral virulence test in mice showed that the virulence of AcrpAcyaAasdC78-1 （pYA3493） was similar with AerpAcyaC78-1, but its median lethal dose is 412 times of vaccine strain C500. Conclusion ： These results demonstrated that attenuated Salmonella choleraesuis AcrpAcyaAasdC78-1 （ pYA3493 ） strain had the potential to be used as an oral live vaccine vector for exoressing foreign genes efficiently.

关 键 词：自杀性质粒 宿主-载体平衡致死系统 ΔcrpΔcyaΔasdC78-1(PYA3493) 口服活疫苗载体 

分 类 号：R378.21[医药卫生—病原生物学]