sH-2K^d-HBc四聚体的构建及其初步的检测应用

Construction and primary application of sH-2K^d-HBc tetramer

目的:sH-2Kd-HBc复合物四聚体的构建与检测,为进一步检测抗原特异性T细胞,探讨免疫发病机制奠定基础。方法:将四个生物素化的可溶性复合物单体与荧光标记的亲和素结合形成四聚体。采用HBcAg真核表达质粒(pcDNA3-C)通过不同的途径免疫小鼠,获得针对核心抗原的特异性CTL,与制备的四聚体共孵育,结合流式细胞仪术进行检测。结果:三种免疫方法所得到的细胞中,抗原特异性CTL频数较对照组都有明显提高(0.24%,0.26%,0.36%vs 0.07%,P≤0.05)。显示制备的四聚体能与抗原特异性T细胞结合,具有检测功能。三种不同的免疫途径所引起的抗原特异性的体液免疫应答和细胞免疫应答强度各有不同。与传统的肌肉注射组相比,基因枪免疫组的体液免疫应答水平较弱而细胞免疫应答水平较强。高压水注射(HDI)组的体液免疫应答和细胞免疫应答水平都要明显高于肌肉注射组结论:获得了功能性的sH-2Kd-HBc复合物四聚体,为进一步检测抗原特异性T细胞奠定基础。

Objective： To prepare and test tetrameric sH-2K^d-HBc complex for the further measurement of the specific CTL response. Methods： PE labled streptavidin with 4 biotinylated binding sites can bind to 4 biotinylatod monomer to form the corresponding tetramer. Mice were immunized via different methods of genetic immunization by use of the construted pcDNA3-C plasmid to get the specific CTLs. Then our prepared tetramer was applied to stain the specific CTLs by the analysis of flow cytometry. Results： We applied our prepared tetramer to stain the cells from the experimental groups and control group. The results showed the tetramer was able to discriminate the frequencies of specific CTL induced by the three immunol methods（0.24 %, 0.26 %, 0.36 % vs 0.07 %, P ≤0.05 ）. This demonstrated that the prepared tetramer could bind its targets specifically and efficiently. The three immunol methods induced different levels of immune responses. Compared with the traditional muscle injection, gene gun induced weaker humoral immune response and stronger cellular immune response, and hydrodynamic in- jection induced the strongest humoral and cellular immune responses. Conclusion： Have successfully constructed the sH-2K^d-HBc tetramcr. The techniques and methods can be used for preparation of tetramers of other types of MHC Ⅰ molecules.