山羊痘口服疫苗构建及其安全性、稳定性检测

Construction,Safety and Stability of Attenuated Salmonella typhimuriumas Delivering Oral Vaccine of Goatpox

目的:探索山羊痘新型疫苗。方法:将山羊痘病毒P32基因插入真核表达载体pcDNA3.1(+),转化至减毒鼠伤寒沙门氏菌SL7207(aroA-);重组菌以不同浓度灌服小鼠进行安全性检测,同时进行稳定性检测。结果:重组减毒沙门氏菌质粒PCR鉴定与预期大小(986bp、1 134bp)一致,完成山羊痘口服疫苗构建;安全性试验结果表明除1010CFU剂量组小鼠呈现轻微反应外,其它组别小鼠均健康存活;体外连续培养10代菌能检测到目的基因,以109CFU剂量灌服小鼠,在第1d、第3d肠内容物和第5d脾脏中分离到重组菌。结论:成功构建了以减毒沙门氏菌为载体的山羊痘口服疫苗,且具有良好的安全性和稳定性,为山羊痘新型疫苗的进一步深入研究奠定了基础。

Objective：Exploring the new vaccine of goatpox.Method：A eukaryotic expression pcDNA3.1（＋）-P32 was constructed by inserting the DNA fragment of Goatpox virus P32 gene.The recombinant plasmid was transformed into an attenuated Salmonella typhimurium SL7207.In safety and stability of the recombinant Salmonella were tested by in vitro and vivo.Result：The plasmid from Salmonella was amplified by PCR with the primers of target gene（986bp） and vector（1134bp）,which constructed the recombinant Salmonella.Experimental groups were orally inoculated with a dose of PBS,108,109,1010CFU per mouse,respectively.There was nothing wrong with the mice of Group PBS,Group 108CFU and Group 109CFU,but Group 1010CFU mice had been diarrheal,and recovered after 3 days.The stability showed that recombinant plasmid can exist stably in vitro 10th generations with selection pressure.The bacteria isolated from the faeces after 1st day,3rd day and spleen after 5th day of mice taken 109CFU inoculation were identified by PCR specific for the target gene and vector.Conclusion：These results indicated that the oral vaccine of goatpox,which possessed relatively safety and stability,was constructed successfully using attenuated Salmonellaas carrier,which provided foundation for further study of the new vaccine of goatpox.