运用体内表达技术筛选霍乱弧菌感染成年小鼠体内诱导表达基因

Use of in vivo expression technology to identify genes preferentially induced during adult mice infection with Vibrio cholerae

为研究霍乱弧菌体内感染机制,寻找新的抗霍乱药物靶标和候选疫苗。以转座子上无启动子的kan-rpsL融合基因作为体内外双重抗生素报告基因,通过与霍乱弧菌埃尔托型C6706接合建立转座子突变库,经体内外各两轮筛选,获得对卡那霉素(Kan)体内抗性体外敏感、链霉素(Str)体外抗性体内敏感的突变株,即转座子插入位点上游包含了能够在体内被诱导激活的启动子。初步建立了筛选霍乱弧菌体内诱导表达基因的成年小鼠模型,最终获得5株阳性克隆。对转座子插入位点进行序列分析,分别为编码渗透敏感型钾离子通道组氨酸激酶的kdpD,谷氨酸合酶大亚基的gltB1,亮氨酰氨基肽酶的pepB以及核苷渗透酶的nupC。对kdpD基因融合lacZ报告基因进行表达调控研究,发现该基因在LB和霍乱毒素诱导培养基AKI中不能被诱导表达。以上结果表明霍乱弧菌通过调节氨基酸及核酸代谢,感知体内外环境来适应宿主体内环境的变化。

To better understand the infection mechanism of Vibrio cholerae and develop new drugs and candidate vaccines for cholera therapy,a novel in vivo expression technology（IVET） was employed to identify in vivo induced（ivi） genes of V.cholerae during adult mice infection.This system employed a kan-rpsL gene fusion as a dual antibiotic selection marker both in vivo and in vitro. The library was pooled by conjugants selected from high concentration of streptomycin,and then subjected to two rounds of in vivo and in vitro selection.Clones harboring an in vivo activated promoter upstream the report genes exhibit kanamycin resistance and streptomycin sensitivity in vivo while kanamycin sensitivity and streptomycin resistance in vitro.Using this strategy,five positive clones,encoding four ivi genes of V.cholerae E1 Tor C6706,have been identified.Bioinformatics analysis showed that these ivi genes involved in environment sensing,amino acid and nucleotide transportation and metabolism.Moreover,the in vitro lacZ reporter gene assay showed that one of the ivi genes,kdpD,had a basal level expression both in LB and AKI medium.All of our findings showed that V.cholerae regulates genes related to environment sensing and metabolism to survive and proliferate in the host.