小肠三叶因子在乳酸菌中的表达

实验目的是在乳酸菌中表达小肠三叶因子(ITF) ,并建立兔子胃溃疡模型,口服观察ITF对胃黏膜损伤的再生作用。在实验中利用了分子克隆技术构建携带ITF基因的重组原核表达质粒 pNICE:sec ITF,将重组质粒转化乳酸菌 NZ9000株 ,筛选鉴定阳性菌落,用nisin诱导表达，表达ITF蛋白通过Tricine SDS-PAGE和 Western blot进行鉴定。将重约2㎏的新西兰成年兔分为对照组,预防组,治疗组,用盐酸诱导胃溃疡模型,预防组在模型建立前用携带 pNICE:sec-ITF的乳酸菌灌胃,对照组,治疗组,在溃疡模型建立后,分别用 PBS、携带pNICE:sec-ITF的乳酸菌灌胃。通过溃疡级别及损伤指数的确定携带 pNICE:sec-ITF的乳酸菌灌胃后对胃黏膜损伤再生的作用。实验成功扩增ITF基因并构建了重组原核表达质粒 pNICE:sec-ITF,转化乳酸菌 NZ9000后经 nisin诱导可表达 Mr约 6.0kDa的重组蛋白 ,表达量约占菌体总蛋白量的 5%。动物实验的预防组和治疗组显示在盐酸诱导胃溃疡模型前和后用携带pNICE:sec-ITF的乳酸菌灌胃，能够促进溃疡黏膜的再生。这对新型的基因工程药物的研究开发具有一定的理论意义，为乳酸菌作为药物递送载体的研究和开发打下一定的实验基础。

Expression of ITF Gene in Lacococcus lactis

To develop an oral drug, ITF gene encoding ITF proein, was expressed  in a live delivery vehicle lactococcus lactis. First, the ITF gene  was cloned into the prokaryotic expressive vector pNICE:sec. Second, the recombinant vector pNICE:sec-ITF was transformated into Lactococcus lactis strain NZ9000 to express ITF protein. Then the recombinant ITF was induced to express and was identified by SDS-PAGE and Western blot.Rabbits are divided into blank control group,preparation group and therapeutic group which are respectively administrated wih PBS and pNICE:sec-ITF Lactococcus lactis. By grades of ulcer test whether administrated pNICE:sec-ITF Lactococcus lactis protects against HCl-induced gastric injury in rabbits. The results were described as follows. The ITF was amplified and cloned in the vector pNICE:sec successfully. The fusion protein (5.9kDa) was expressed in L. lactis by the induction of the nisin. The quantity of expression accounted for 5% of the total bacterial protein. Western bolt analysis confirmed that fusion protein could be recognized specially by Monoclonal Anti-human TFF3 Antibody. Preparation groups and therapeutic groups do good than control group. Prove that administrated pNICE:sec-ITF Lactococcus lactis is biologically active in an HCl-induced rabbit gastric mucosal injury model.