稳定携带TAT-Apoptin融合基因的重组减毒鼠伤寒沙门菌的构建及其基本生物学特性的测定

Construction of recombinant attenuated Salmonella enterica serovar Typhimurium stably harbouring TAT-Apoptin fusion gene and determination of biological characteristics of the strain

为构建能稳定携带TAT-Apoptin融合基因的重组减毒鼠伤寒沙门菌,以pMD19-T-Apoptin为模板,PCR扩增出TAT-Apoptin融合基因,将其连接至pYA3493,构建了重组质粒pYA3493-TAT-Apoptin;然后将其电转至减毒鼠伤寒沙门菌SL1344ΔcrpΔasd,并对该重组减毒沙门菌的生长特性、遗传稳定性和表达特性进行了测定。PCR和测序结果表明,成功构建了重组质粒pYA3493-TAT-Apoptin。进一步对重组减毒鼠伤寒沙门菌SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)研究发现,其生长速度比强毒株SL1344明显减慢,与缺失株SL1344ΔcrpΔasd及互补菌株SL1344ΔcrpΔasd(pYA3493)基本一致,且能够稳定遗传TAT-Apoptin融合基因。重组菌培养上清经SDS-PAGE能检测到TAT-Apoptin蛋白条带。以上结果证明,能稳定携带TAT-Apoptin融合基因的重组减毒鼠伤寒沙门菌SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)构建成功,且该重组菌能分泌性表达TAT-Apoptin蛋白。

This study was aimed to construct a recombinant attenuated Salmonella enterica serovar Typhimurium strain for stable delivery of TAT-Apoptin fusion gene.Firstly,the TAT-Apoptin fusion gene was amplified from the recombinant plasmid pMD19-T-Apoptin by PCR,and inserted into the vector pYA3493.The recombinant plasmid pYA3493-TAT-Apoptin was electro-transformed into the attenuated S.entericaserovar Typhimurium SL1344ΔcrpΔasd.Then the growth characteristics,genetic stability and expression property of the recombinant attenuated S.enterica serovar Typhimurium SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)were analyzed.The results of PCR and sequencing showed that recombinant plasmid pYA3493-TAT-Apoptin was successfully constructed.The growth rate of recombinant attenuated S.entericaserovar Typhimurium SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)strain was more slowly than the SL1344 strain,but almost identical to SL1344ΔcrpΔasdstrain and SL1344ΔcrpΔasd(pYA3493)strain.The TAT-Apoptin fusion gene was inherited stably and TAT-Apoptin protein was detected in the cultured supernatant of SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)strain.These results showed that the recombinant attenuated S.enterica serovar Typhimurium SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)strain was successfully constructed and TAT-Apoptin protein was expressed in the form of secretion.