CAJ | 학술논문

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。
목적：구건병감정휴대인IL-10기인적대장간균-쌍기간균천사분비형표체재체，탐색기인치료적신형재체。방법선택이왕시험이구건호적질립재체pBADs-GFP여생물합성적함유hIL-10질립，통과쌍매절、매련반응구건병감정 pBADs-hIL-10천사질립，장 pBADs／hIL-10질립전전화입장쌍기간균（NCC 2705），합성BL-hIL-10，사선출양성극륭균주，용RT-PCR법험증질립hIL-10적기인표체후，이0．2％ L-아랍백당（L-Arb）유도표체12 h、24 h、36 h후，분별용ELISA화Western blot법험증전기인쌍기간균중hIL-10단백표체수평，병선택최가적유도표체시간。결과재장쌍기간균NCC 2705중，성공적구건료능분비hIL-10단백적BL-hIL-10전체체；기최가유도적표체시간시24 h。결론재장쌍기간균（NCC 2705）내성공구건료일충신적、유효적IL-10적전체계통，BL-hIL-10능은정표체구유생물활성적hIL-10단백。위IL-10전기인쌍기간균치료IBS적적연구전정료기출。
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.