携带Apoptin的减毒鼠伤寒沙门菌Ⅲ型分泌系统的构建

Construction of recombinant attenuated Salmonella typhimurium harbouring apoptin gene by type Ⅲ secretion system

为研究减毒鼠伤寒沙门菌Ⅲ型分泌系统介导Apoptin的特性,将Apoptin基因克隆入载体pYA3493-SopE,构建重组载体pYA3493-SopE-Apoptin,然后将其电转化至减毒鼠伤寒沙门菌ΔcrpΔasd SL1344,对该重组菌生长特性、生化特性、毒力和表达等特性进行分析;同时将重组菌体外感染B16F10黑色素瘤细胞,分析其介导Apoptin对B16F10的作用。PCR、酶切检测表明,携带Apoptin的重组减毒鼠伤寒沙门菌ΔcrpΔasd SL1344(pYA3493-SopE-Apoptin)构建成功;生物学特性分析发现,重组菌的生长特性、生化特性与缺失株ΔcrpΔasd SL1344和互补株ΔcrpΔasd SL1344(pYA3493-SopE)相近,与亲本株SL1344差异明显;腹腔感染重组菌的C57BL/6小鼠的LD50为1.69×10^7CFU;在重组菌培养上清和感染的B16F10细胞内均可检测到SopE-Apoptin蛋白条带。进一步研究其介导Apoptin对B16F10细胞的作用发现,重组菌可抑制该细胞增殖,且具有MOI浓度梯度和感染时间依赖性;用TUNEL法可观察到TUNEL阳性细胞;Western-blot可在重组菌感染的细胞内检测到Caspase-9凋亡信号蛋白表达。以上结果表明,减毒鼠伤寒沙门菌Ⅲ型分泌系统能够介导Apoptin蛋白的分泌性表达,且能够诱导肿瘤细胞B16F10的凋亡。

This study was aimed to explore the characteristics of attenuated Salmonella typhimurium ΔcrpΔasdSL1344(pYA3493-SopE) harbouring Apoptin gene by type Ⅲ secretion system.Firstly,the Apoptin gene was subcloned into the vector pYA3493-SopE and the recombinant plasmid pYA3493-SopE-Apoptin was electro-transformed into attenuated S. typhimurium ΔcrpΔasd SL1344.Then,the growth characteristics,biochemical characteristics,virulence and expression property of the recombinant strain ΔcrpΔasd SL1344(pYA3493-SopEApoptin) were analysed.The effects on the B16F10 melanoma cells infected by recombinant strain were observed.The results of PCR and enzyme digestion showed that the recombinant attenuated S. typhimurium ΔcrpΔasdSL1344(pYA3493-SopE-Apoptin) was constructed successfully.The biological characteristics showed that the growth and biochemical characteristics of the recombinant strain were similar to the strains ΔcrpΔasd SL1344 andΔcrpΔasd SL1344(pYA3493-SopE),but significantly different from the virulent strain SL1344.The LD50 of the recombinant strain infected with C57BL/6 mice by intraperitoneal injection was 1.69 ×10^7 CFU.SopE-Apoptin protein bands were been detected in the cultured supernatant and the infected B16F10 cells of recombinant strain.Furthermore,the results indicated that the recombinant strain inhibited the proliferation of B16F10 cells in a MOI concentration and time dependent manner.TUNEL positive cells were observed.Apoptosis signaling protein Caspase-9 was detected in recombinant strain-infected B16F10 cells by Western-blot.These results demonstrated that the Apoptin mediated by the attenuated S. typhimurium type Ⅲ secretion system were expressed in the form of secretion,and induced the apoptosis of B16F10 melanoma cells.