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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: To develop an oral live vaccine vector that can stably carry heterologous genes in swine. Methods: Based on the ΔcrpΔcya C78-1 attenuated Salmonella typhimurium, the recombinant plasmid pREasd-mediated bacterial homologous recombination was used to construct the deletion mutant ΔcrpΔcyaΔasd C78-1, and then the complementary plasmid p YA3493 was electroporated into the above strain to construct a host-vector balance lethal system of ΔcrpΔcyaΔasd C78-1 (p YA3493) and to further investigate its biological characteristics. Results: PCR and sequencing results together showed that the construction of ΔcrpΔcyaΔasd C78-1 (pYA3493) was successful. The results of biological tests showed that the serotype of ΔcrpΔcyaΔasd C78-1 (pYA3493) was the same as the parent strain ΔcrpΔcya C78-1 and the vaccine strain C500, and was able to stabilize the genetically deficient asd gene fragment; its growth rate was slightly slower than that of ΔcrpΔcya C78-1 , And both were significantly slower than vaccine strain C500; their biochemical properties were also substantially the same as for ΔcrpΔcya C78-1. The oral challenge test in mice showed that the virulence of ΔcrpΔcyaΔasd C78-1 (pYA3493) was almost equivalent to ΔcrpΔcya C78-1, but its half lethality was about 412 times that of the vaccine strain C500. Conclusion: The above results show that attenuated Salmonella choleraesuis ΔcrpΔcyaΔasd C78-1 (p YA3493) has the potential as an oral live vaccine vector for efficient expression of foreign genes.