空肠弯曲菌外膜蛋白PEB1介导的重组BCG与上皮细胞结合能力的研究

发布时间：2018-05-25 19:57

本文选题：重组BCG + 细胞粘附　；参考：《第四军医大学》2011年硕士论文

【摘要】：背景疫苗在预防和治疗人类多种疫症的过程中发挥了不可磨灭的作用。其中,基因工程重组活载体疫苗因其能够诱导产生体液、细胞甚至黏膜免疫而受到广大研究者的关注。基因重组卡介苗(Gene recombinant Bacillus Calmette-Guerin vaccine,rBCG)就是一种单次接种即可持续诱导机体对靶抗原的免疫应答的基因工程疫苗。它的构建是利用基因工程学技术对BCG基因进行改造使其提高免疫原性,诱导产生对一种或多种疾病的持久防御,以预防和治疗疾病。迄今为止,研究者们已成功制备了多种携带不同抗原的rBCG,并将其用于结核[1]、肿瘤[2]和寄生虫感染[3]等多种疾病的防治。本研究小组前期已成功构建胞壁型Der p2-rBCG和胞壁型PEB1-Der p2-rBCG的基因重组疫苗。在此研究的基础上,我们采用体外细胞培养的方法,评价疫苗PEB1-Der p2-rBCG与上皮细胞的结合能力。目的评价PEB1介导的屋尘螨抗原(Der p2)重组BCG疫苗(PEB1-Derp2-rBCG)与人上皮细胞的结合能力。实验方法和结果 1.胞壁型PEB1-Der p2-rBCG与上皮细胞的粘附实验采用体外细胞培养的方法,分别将普通BCG、胞壁型Der p2-rBCG和胞壁型融合蛋白PEB1-Der p2-rBCG与HeLa细胞及人类肠黏膜上皮细胞(HIEC)进行共孵育,利用HE和抗酸染色法对各组细胞与疫苗的粘附结果进行染色,光学显微镜下计数各组的粘附率,并进行比较。孵育24小时后,无论HeLa细胞还是HIEC,PEB1-Der p2-rBCG组较普通BCG组和Der p2-rBCG组的粘附率明显提高,差异有显著性(P0.05),但是,Der p2-rBCG组与普通BCG组比较没有明显差异(P0.05)。 2.胞壁型PEB1-Der p2-rBCG与上皮细胞的抑制和阻断实验采用相同的方法将各组疫苗与细胞进行共孵育,并对各组分别加入PEB1蛋白或抗PEB1多克隆抗体进行粘附抑制和阻断,于相同的时间点终止孵育染色后观察其对结合能力的影响。结果显示PEB1蛋白或抗PEB1多克隆抗体的加入对PEB1-Der p2-rBCG组的粘附功能有明显抑制作用(两组都P0.05);但是,对普通BCG组和Der p2-rBCG组的粘附无影响(各组间P0.05)。结论空肠弯曲菌外膜蛋白PEB1具有介导增强PEB1-Der p2-rBCG与上皮细胞粘附的能力。
[Abstract]:Background Vaccines play an indelible role in the prevention and treatment of human diseases. Among them, recombinant vector vaccine has attracted much attention because of its ability to induce humoral, cellular and even mucosal immunity. Recombinant Bacillus Calmette Calmette Guerin (BCG) Gene recombinant Bacillus Calmette-Guerin Vaccine (rBCG) is a kind of genetically engineered vaccine which can continuously induce immune response to target antigen by single inoculation. Its construction is to improve the immunogenicity of BCG gene by genetic engineering technology, and to induce a lasting defense against one or more diseases in order to prevent and treat the disease. So far, researchers have successfully prepared a variety of rBCGs carrying different antigens and used them in the prevention and treatment of tuberculosis [1], tumor [2] and parasitic infection [3]. In the early stage of this study, we have successfully constructed the recombinant vaccine of cell wall type Der p2-rBCG and cell wall type PEB1-Der p2-rBCG. On the basis of this study, we used the method of cell culture in vitro to evaluate the binding ability of vaccine PEB1-Der p2-rBCG to epithelial cells. Purpose To evaluate the binding ability of PEB1 mediated Der p2) recombinant BCG vaccine PEB1-Derp2-rBCGwith human epithelial cells. Experimental methods and results 1. Adhesion of cell wall type PEB1-Der p2-rBCG to epithelial cells Common BCG, cell wall type Der p2-rBCG and cell wall type fusion protein PEB1-Der p2-rBCG were incubated with HeLa cells and human intestinal epithelial cells by cell culture in vitro. HE and acid fast staining were used to stain the adhesion of cells to the vaccine. The adhesion rates of each group were counted under optical microscope and compared. After incubation for 24 hours, the adhesion rate of HeLa cells and HIEC-PEB1-Der p2-rBCG group was significantly higher than that of normal BCG group and Der p2-rBCG group, and the difference was significant (P 0.05), but there was no significant difference between Der p2-rBCG group and BCG group. 2. Inhibition and blocking of cell wall type PEB1-Der p2-rBCG and epithelial cells The same method was used to incubate each vaccine with the cells, and the adhesion inhibition and blocking of each group with PEB1 protein or anti PEB1 polyclonal antibody were carried out, and the effect on binding ability was observed after the incubation staining was terminated at the same time point. The results showed that the addition of PEB1 protein or anti PEB1 polyclonal antibody could significantly inhibit the adhesion function of PEB1-Der p2-rBCG group (P0.05 in both groups, but had no effect on the adhesion between normal BCG group and Der p2-rBCG group (P0.05). Conclusion Campylobacter jejuni outer membrane protein PEB1 can mediate the enhancement of PEB1-Der p2-rBCG adhesion to epithelial cells.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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