SG97A平衡致死系统的构建及其初步应用

Construction and Preliminary Application of Balanced Lethal System Based on Attenuated Salmonella SG97A

敲除鸡伤寒沙门菌(SG)减毒疫苗株97Aasd基因,构建宿主载体平衡致死系统并表达新城疫病毒(NDV)F蛋白,构建二价活菌苗。PCR扩增两段位于asd基因外侧的序列作为等位基因,以氯霉素抗性(CmR)基因替代asd基因,构建自杀质粒pGMB151Δasd::CmR,通过E.coliχ7213与SG97A固相杂交,将质粒转入SG97A,反向筛选获得SG97AΔasd::CmR,利用质粒pCP20去除CmR后,DAP的依赖菌株命名为SG97AΔasd。将含asd基因的质粒pYA3334转入SG97AΔasd中构建宿主载体平衡致死系统,并表达NDV-F蛋白。结果表明,通过等位基因重组方法,成功构建SG97AΔasd及宿主载体平衡致死系统,重组菌能够表达NDV-F蛋白,并能在鸡体内诱导产生特异性抗体。本研究成功构建SG97AΔasd宿主载体平衡致死系统,并表达NDV-F蛋白及其诱导宿主产生抗体,为应用该系统构建二价活菌苗的研究奠定了基础。

The aim of this study was to delete asdgene of Salmonella gallinarum(SG)attenuated vaccine strain 97 Aand construct bivalent live vaccine based on host-vector balanced lethal system to express F protein of Newcastle disease virus(NDV).Two DNA fragments flanking asd gene as allele were amplificated by PCR and asdgene was replaced by chloramphenicol resistance(CmR)gene to construct the suicide plasmid pGMB151Δasd::CmR.After conjugation,the recombiant plasmid was transformed into SG97 Afrom E.coliχ7213,SG97AΔasd::CmR was reversely screened out under the selectable pressure of chloramphenicol,DAP-dependent strain was named SG97AΔasd.Vector-host balanced lethal system was construct by pYA3334 containing asdgene being transferred into SG97AΔasdand to expressed F protein of NDV.SG97AΔasd was successfully constructed by allelic exchanges,its growth was DAP-dependent.Vector-host balanced lethal system was constructed by pYA3334 being transferred to SG97AΔasd and could express F protein of NDV and also induce specific antibodies in infected chickens.Vector-host balanced lethal system was constructed successfully based on attenuated Salmonella SG97AΔasdin this study and could express Fgene of NDV as foreign gene to induce special antibodies in animal model,it laidafoundation for bivalent live vaccine based on attenuated Salmonella.