重组乳链菌介导肠上皮细胞表达抗TNF-α抗体对实验性结肠炎治疗的实验研究

Background/Aims. The pathogenesis of inflammatory bowel disease (IBD) is largely unknown, though it is believed that cytokines and pre-inflammatory factors play important role in it. Recent years, the monoclonal antibody against tumor necrosis factor-alpha (TNF-alpha) has been used for IBD therapy and achieved successfully. The aim of our study is to express and secret anti-TNF-alpha antibody in intestinal epithelium mediated by genetically modified food-grad Lactococculs lactis ( L. lactis) for treatment of experimental colitis in mice. Approach:. To facilitate gut epithelial cell to express and secrete scFV-TNF alpha, we first construct a bacteria-mediated trans-kingdom system, in which the invasin gene (inv) of Yersinia pseudotuberculosis and listeriolysin O gene (hly) from Listeria monocytogenes are introduced into a E. coli - L. latis shuttle vector. The recombinant L. lactis transformed with the bacteria-mediated trans-kingdom vector can act as DNA delivery systems into mammalian cells through the following mechanisms: at first, engineered bacteria expressing invasin are able to enter into mammalian cells by binding integrins. And then, the bacteria are lysed and released plasmid DNAs to the cytosol while listeriolysin is active at low pH,as in phagolysosomes. . In the experiment, The single-chain variable fragment antibody against TNF-alpha (scFV-TNF-alpha) is used. The DNA sequence of scFV-TNF-alpha is synthesized by gene assembly method using oligodeoxyribouncleotieds in vitro. For expression of scFV-TNF-alpha, we built a eukaryotic cell gene expression unite, that is, scFV-TNF-alpha with signal peptide from tac antigen is driven by chicken β-actin promoter which followed by poly (A) sequence. The scFV-TNF-alpha eukaryotic cell gene expression unite is further introduced into the bacteria-mediated trans-kingdom system. After L. lactis is transformed with the tans-kingdom vector, the recombinant bacteria can invade and deliver plasmids containing scFV-TNF-alpha expression unite into gut epithelial cells so that scFV-TNF-alpha can be expressed and secreted into gut in vivo. . To verify therapeutic efficacy of scFV-TNF-alpha recombinant L. lactis on colitis, the experimental colitis model are induced in mice by 2,4-Dinitrochlorobenzene (DNCB) and Trinitrobenzenesulfonic acid (TNBS) separately. After mice fed with the recombinant L. lactis, the viability of strain and the amount of svFC-TNF-alpha in gut will be measured, the injury index of colitis, survival rate of mice will be observed. . Expected Results: . We expected the bacteria-mediated trans-kingdom system will help to express foreign DNA in gut mucosal. The scFV-TNF-alpha expressed by recombinant bacteria in epithelial cells could heal experimental colitis.

本研究拟用食品级乳酸链球菌（乳链菌）介导肠道上皮细胞表达抗肿瘤坏死因子单链抗体（scFv-TNF-α）,用于实验性结肠炎的治疗。研究中采用基因拼装的方法,体外合成scFv-TNF-α。将假结核耶尔森菌的侵袭素（Inv）和单核细胞增多性李斯特菌的溶血素(hly)基因,克隆于乳链菌质粒并转化乳链菌,使重组乳链菌不仅可侵入非吞噬性哺乳动物细胞,还能使细菌从细胞进入囊泡中逃逸从而使细菌的质粒从空泡释放至细胞胞浆中。同时,我们还将在质粒的多克隆位点插入真核细胞表达元件和scFv-TNF-α序列,以期可以在细胞中转录、表达和分泌scFv-TNF-α。最后,将建立小鼠实验性结肠炎模型,用建立的跨界重组乳链菌灌胃以期使乳链菌介导小鼠肠上皮表达scFv-TNF-α,观察细菌介导肠上皮表达scFv-TNF-α对实验性结肠炎的治疗作用。将探索建立一种可能用口服细菌介导表达抗TNF-α抗体治疗炎症性肠病的方法。

本课题旨在构建的跨界乳链菌表达scFv-TNF-α抗体用于炎症性肠病的治疗。我们把“菌感”必需的Inv及hly基因通过同源重组与乳链菌基因组上的thyA基因进行基因置换，构建具有更高安全性的thyA缺陷的跨界乳链菌表达系统。把PEG-3启动子、目的表达基因及真核细胞的表达元件等则克隆于乳链菌穿梭质粒pTRKH2中，这样就可根据实际情况加入不同的治疗基因。所以本课题构建的跨界乳链菌不单可以用于炎症性肠病的治疗，更多的价值还在于探索一种崭新的口服乳链菌介导基因治疗的给药途径。