减毒猪霍乱沙门菌△cya△asdC78-1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 △cya△asdC78-1

为研制能够稳定携带外源基因的猪霍乱沙门菌口服活疫苗载体,本研究在减毒猪霍乱沙门菌△cyaC78-1基础上,利用自杀性质粒介导的等位交换技术,构建了△cya△asdC78-1宿主-载体平衡致死系统,并对其生物学特性进行了鉴定。PCR及测序结果表明△cya△asdC78-1构建正确,其生物学特性检测结果显示△cya△asdC78-1生化特性与△cyaC78-1完全相同,血清型与△cyaC78-1和疫苗株C500相同,并且具有稳定的遗传特性;其生长速度与△cya C78-1基本相同,均明显慢于疫苗株C500。动物试验表明△cya△asdC78-1电转化携带asd基因的互补质粒pYA3493后,其毒力比△crp△asdC78-1(p YA3493)降低1.14倍,比△asdC500(pYA3493)降低了5.1倍。以上结果表明,减毒猪霍乱沙门菌△cya△asdC78-1同样可以作为口服活疫苗载体用于高效表达外源基因,为开发以C78-1为基础的口服多价疫苗载体奠定了基础。

Abstract： To develop an oral live vaccine vector of Salmonella choleraesuis for swine which was able to stably carry exogenous genes, the S.choleraesuis △ cya △ asdC78-1 host-vector balanced lethal system was constructed with the method of recombinant suicide plasmid-mediated allelic exchange on the basis of attenuated S.choleraesuis △ cyaC78-1, and the biological characteristics of △ cya △asdC78-1 showed that the biochemical characteristics and the serotype of A cya A asdC78-1 were exactly the same with △cyaC78-1 strain; The growth rate of A cyaA asdC78-1 was basically the same as △cyaC78-1 strain, but both of their growth speeds were significantly slower than vaccine strain C500. Animal experiment showed that the virulence of △ cya ZkasdC78-1 carried complementary plasmid pYA3493 by electroporation was decreased by 1.14 and 5.1 times compared with △ crp △asd C78-1 （pYA3493） and △ asdC500 （pYA3493） in mice. In conclusion, these results demonstrated that attenuated S. choleraesuis △ cya △ asdC78-1 could be used as an oral live vaccine vector for expressing foreign genes efficiently and facilitate for developing an oral polyvalent vaccine vector based on the C78-1 strain.