表达NDV HN蛋白重组减毒鸡白痢沙门菌ΔcrpΔasdC79-13的构建及生物学特性  

Construction and biological characteristics of recombinant attenuated Salmonella pullorum ΔcrpΔasdC79-13 expressing HN antigen of Newcastle disease virus

作　　者：尚珂[1] 程相朝[1] 郁川[1] 余祖华[1] 张俊峰[1] 毛福超 韩璐[1] 韩于佩 刘文慧[1] 陈桂华[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,河南洛阳471003

出　　处：《中国兽医学报》2016年第4期589-594,共6页Chinese Journal of Veterinary Science

基　　金：河南省科技攻关资助项目(112102110019)

摘　　要：利用平衡致死系统构建表达鸡新城疫病毒(newcastle disease virus,NDV)血凝素-神经氨酸酶(HN)的减毒鸡白痢沙门菌活载体疫苗株。以p MD18-T-HN为模板,经PCR扩增出HN基因,定向插入原核表达载体p YA3493,构建重组质粒p YA-HN,再将其电转入减毒鸡白痢沙门菌ΔcrpΔasd C79-13中,获得重组减毒鸡白痢沙门菌ΔcrpΔasd C79-13(p YA-HN)。对重组菌的生化特性、生长特性、稳定性及安全性进行测定;并对重组菌表达的HN蛋白进行SDSPAGE和Western blot分析。结果显示,该重组菌保留了在DAP阴性环境中的生存能力;不能利用麦芽糖、蔗糖及乳糖等碳源,但保留了利用葡萄糖的能力;其生长速度明显低于强毒株C79-13,而与ΔcrpΔasd C79-13相比变化不明显;在体外连续传代能稳定遗传HN基因片段;经SDS-PAGE出现1条蛋白质量约为63 000的蛋白条带;Western blot证明重组菌表达的HN蛋白能与NDV阳性血清特异性结合;雏鸡口服接种试验表明ΔcrpΔasd C79-13(p YA-HN)的毒力较强毒株C79-13下降183倍。上述结果表明,成功构建了能稳定表达NDV HN蛋白的口服重组减毒鸡白痢沙门菌,为开发鸡白痢—新城疫的基因工程口服疫苗奠定了初步基础。In order to construct recombinant attenuated Salmonella enterica serovar pullorum oral live vector strain expressing HN antigen of Newcastle disease （ND）. The HN gene was cloned from the plasmid pMD-18T-HN by PCR and inserted into the vector pYA3493 to construct recobi- nant plasmid pYA-HN. The recombinant plasmid pYA-HN was finally electro-transformed into attenuated Salmonella enterica serovar pullorum ΔcrpΔasdC79-13. The recombinant strain ΔcrpΔasdC79-13 （pYA-HN） was identified with biochemical traits, stability, growth curve and safety in vitro ,and its HN protein expression was analyzed by SDS-PAGE and Western blot. The growth ability of recombinant stain ΔcrpΔasdC79-13 （pYA-HN） in a circumstance without DAP was persisted ; The ability of fermentation for maltose, sucrose, and lactose was lost, but the recom- binant could ferment glucose. Compared with C79-13 strain,the growth velocity was reduced sig- nificantly but the same as ΔcrpΔasdC79-13. The gene of HN can be transmitted stably in the liquid nutrient medium of luria bertani after 60 generations;furthermore,the SDS-PAGE results showed a protein band with molecular weight of 63 000. Western blot confirmed the HN protein expressed by the recombinant could bind specifically with NDV positive serum,the chicken oral innoculation test showed that the virulence of the Acrp2xasdC79-13 （pYA-HN） recombinant was lower 183 times than C79-13. A recombinant attenuated Salmonella pullorum C79-13 strain expressing HN antigen of Newcastle disease virus was constructed successfully which lays a foundation for develo- ping genetically oral vaccine of pullorum disease-newcastle disease.

关 键 词：asd^+宿主—载体平衡致死系统 减毒鸡白痢沙门菌 NDV HN 生物学特性 

分 类 号：S852.65[农业科学—基础兽医学]