携带IL-10基因双歧杆菌载体的构建及鉴定  

Construction and identification Bifidobacterium as a delivery system of IL -10

作　　者：张定国[1] 姚君[1] 朱忠生[2] 赖明广[1] 魏铖 王立生[1] 

机构地区：[1]深圳市人民医院消化内科暨南大学第二临床医学院消化内科,广东省深圳518020 [2]深圳市儿童医院消化内科

出　　处：《中国基层医药》2015年第8期1136-1138,共3页Chinese Journal of Primary Medicine and Pharmacy

基　　金：广东省深圳市医疗卫生类科研项目（201302015）;深圳市科技技术创新委员会基础研究项目（JCYJ20130402092657774）

摘　　要：目的：构建并鉴定携带人IL-10基因的大肠杆菌-双歧杆菌穿梭分泌型表达载体，探索基因治疗的新型载体。方法选择以往试验已构建好的质粒载体pBADs-GFP与生物合成的含有hIL-10质粒，通过双酶切、酶连反应构建并鉴定 pBADs-hIL-10穿梭质粒，将 pBADs／hIL-10质粒电转化入长双歧杆菌（NCC 2705），合成BL-hIL-10，筛选出阳性克隆菌株，用RT-PCR法验证质粒hIL-10的基因表达后，以0．2％ L-阿拉伯糖（L-Arb）诱导表达12 h、24 h、36 h后，分别用ELISA和Western blot法验证转基因双歧杆菌中hIL-10蛋白表达水平，并选择最佳的诱导表达时间。结果在长双歧杆菌NCC 2705中，成功的构建了能分泌hIL-10蛋白的BL-hIL-10传递体；其最佳诱导的表达时间是24 h。结论在长双歧杆菌（NCC 2705）内成功构建了一种新的、有效的IL-10的传递系统，BL-hIL-10能稳定表达具有生物活性的hIL-10蛋白。为IL-10转基因双歧杆菌治疗IBS的的研究奠定了基础。Objective To construct and identify a novel IL-10 delivery system by transforming a hIL-10-containing plasmid into B.longum （BL -hIL -10）.Methods A plasmid vector pBADs -GFP was selected which had been built by previous test and biosynthetic hIL-10 plasmid,through double enzyme digestion and enzyme reaction,to construct and identify PBADs-hIL-10 shuttle plasmid,then to synthesis BL-hIL-10.hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after 0.2% L-arabinose induction in vitro as examined by Western blot,enzyme-linked immunosorbent assay （ELISA）and RT-PCR;Culture supernatants and bacterium pellets were collected after continuous culture for 12,24 and 36h,respectively.hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after 0.2% L-arabinose induction in vitro as examined by Western blot,enzyme -linked immunosorbent assay （ELISA）and RT -PCR;Culture supernatants and bacterium pellets were collected after continuous culture for 12,24 and 36h,respectively.Results The BL-hIL-10 bacterial strain that can stably express hIL-10 factor was successfully screened out,and the levels of hIL-10 in both superna-tant and cell pellet were similarly reached maximum at 24h of culture.Conclusion BL-hIL-10 as a novel oral hIL-10 delivery system has been successfully established,which established a basis for the treatment of IBS with transgenic Bifidobacterium.

关 键 词：长双歧杆菌 IL-10 穿梭质粒 构建 鉴定 

分 类 号：R346[医药卫生—基础医学]