重组乳链菌介导肠上皮细胞表达及分泌抗肿瘤坏死因子单克隆抗体

Anti-TNF－α monoclonal antibodies is an effective biological agent in the treatment of inflammatory bowel disease,but the price of this agent is expensive .Using it frequently may produce anti-TNF-α mirror antibody .On the base of our experiments supported by a previous NSFC programme(NO.81250012), we have replaced the thymidylate synthase in Lactococcus lactis genome with the Yersinia pseudotuberculosis invasin (inv) and mononucleosis Lester bacteria hemolysin (hly) gene.And we make a thyA gene deficient recombinant Lactococcus lactis .We will electrotransform the thyA gene deficient recombinant Lactococcus lactis with the pladmid which has anti-TNF－αmonoclonal antibodies sequences , eukaryotic expression element,polyA sequences. Lactococcus lactis can invade the intestinal epithelial cells and release the plasmid into the cytosol .The intestinal epithelial cells will transcribe and translate the eukaryotic expression element sequences of the plasmid . Therefore ,the intestinal epithelial cells can express and secrete anti-TNF-α monoclonal antibody . The efficiency of monoclonal antibody expression will be evaluated by in vitro cell culture and in vivo animal experiments . The thyA deficient Lactococcus lactis has no biological pollution, because it could not survive in the environment.Moreover,the approach of experiment is convenient.Anti-TNF-α monoclonal antibody is produced by the host ,so it can effectively reduce the incidence of mirror antibody .

抗TNF－α单克隆抗体是IBD治疗的有效生物制剂，但价格昂贵，多次使用甚至产生抗TNF－α镜像抗体影响疗效。在前一年短期国家自然基金研究的基础上，我们将以同源重组的方法，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，取代乳链菌基因组中的胸苷酸合成酶（thymidylate synthase，ThyA）基因，制备thyA基因缺陷型重组乳链菌。然后，构建含全长抗TNF-α单抗真核元件的乳链菌质粒，转化thyA基因缺陷型乳链菌。得到的重组乳链菌可侵袭肠黏膜上皮细胞，并在细胞内释放质粒。细胞通过转录质粒载体的真核表达元件，表达和分泌抗人TNF-α单克隆抗体。最后通过体内细胞培养和动物试验评价单抗表达效率。由于thyA缺陷型乳链菌在体外的环境不能生存，对环境将无生物污染。本研究细菌制备简便；单抗是由宿主产生的，将可有效避免抗抗体的产生。

近年来，生物制剂在临床治疗炎症性肠病中发挥重要的作用，尤其对于一些氨基水杨酸、糖皮质激素、免疫抑制治疗无效的患者也有较好的治疗效果。目前，临床上最常用的生物制剂是抗肿瘤坏死因子-α抗体，如英夫利昔单抗。本项目利用基因拼装技术合成人源化抗人TNF-α单抗的重链和轻链可变区序列，通过融合PCR方法克隆全长抗人TNF-α单抗。抗体重链和轻链之间含有Furin酶裂解序列和手足口病病毒衍生的2A裂解序列，保证重链和轻链按1:1比例组装。我们将利用乳链菌-大肠杆菌载体构建完整的抗人TNF-α单抗真核表达载体。通过同源重组方法将乳链菌ThyA基因替换为inv、hly基因，制备缺陷型ThyA乳链菌。通过“菌感”方法，利用缺陷型ThyA乳链菌使质粒在细胞内表达抗人TNF-α抗体，用于治疗炎症性肠病。ThyA缺陷型乳链菌不能在外界环境生存，具有一定的生物安全性。重组菌制备成本低，能降低患者治疗费用，使患者长期有效接受抗TNF-α单抗治疗。

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