Wiest-Ladenburger U, Fortnagel A, Richter W, Reimann J, Boehm B O
Department of Internal Medicine I, University of Ulm, Germany.
Horm Metab Res. 1998 Oct;30(10):605-9. doi: 10.1055/s-2007-978942.
The technique of DNA-based vaccination was used to generate a T-cell-dependent antibody response to glutamic acid decarboxylase (GAD) in BALB/c, C57BL/6, and non-obese diabetic (NOD) mice. Plasmids were constructed in which the expression of the rat GAD65 (rGAD65) or the rat GAD67 (rGAD67) gene was driven by the immediate early region promoter of the human cytomegalovirus (pCMV). This "naked" plasmid DNA was then injected into the regenerating muscles of the studied mice. In the vaccinated animals, antibody responses to GAD65 or to GAD67 were induced. Epitope recognition of GAD was studied by protein footprinting, a technique which makes use of a limited proteolysis of antibody-bound antigen. Different epitope recognition patterns were found, corresponding to strain-specific patterns. Mild trypsin treatment generated 50 kD, 46 kD, 40 kD, 30 kD, and 21 kD proteolytic fragments. In NOD mice, 50, 46 and 40 kD bands were the most prominent signals. In non-diabetes prone BALB/c mice, a faint 40 kD band appeared suggesting a rather weak protection of GAD from tryptic lysis. The pattern observed in C57BL/6 mice was more comparable to the NOD mice pattern with prominent 40 kD and 30 kD signals and a faint 21 kD fragment. Diabetes incidence was unchanged in NOD mice, and no diabetes was observed in C57BL/6 and BALB/c mice, respectively. The data demonstrate that genetic immunization is a suitable novel tool to stimulate and to manipulate an immune response against the diabetes-associated protein glutamic acid decarboxylase. Interestingly, our results indicate that, by genetic vaccination, distinct B-cell epitopes were generated in the various studied mouse strains.
采用基于DNA的疫苗接种技术，在BALB/c、C57BL/6和非肥胖糖尿病（NOD）小鼠中产生针对谷氨酸脱羧酶（GAD）的T细胞依赖性抗体反应。构建了质粒，其中大鼠GAD65（rGAD65）或大鼠GAD67（rGAD67）基因的表达由人巨细胞病毒的立即早期区域启动子（pCMV）驱动。然后将这种“裸”质粒DNA注射到受试小鼠的再生肌肉中。在接种疫苗的动物中，诱导了对GAD65或GAD67的抗体反应。通过蛋白质足迹法研究了GAD的表位识别，该技术利用抗体结合抗原的有限蛋白酶解。发现了不同的表位识别模式，对应于品系特异性模式。温和的胰蛋白酶处理产生了50 kD、46 kD、40 kD、30 kD和21 kD的蛋白水解片段。在NOD小鼠中，50、46和40 kD条带是最突出的信号。在非糖尿病易感的BALB/c小鼠中，出现了一条微弱的40 kD条带，表明GAD对胰蛋白酶裂解的保护作用较弱。在C57BL/6小鼠中观察到的模式与NOD小鼠模式更相似，突出的40 kD和30 kD信号以及一条微弱的21 kD片段。NOD小鼠的糖尿病发病率没有变化，C57BL/6和BALB/c小鼠分别未观察到糖尿病。数据表明，基因免疫是一种合适的新型工具，可刺激和操纵针对糖尿病相关蛋白谷氨酸脱羧酶的免疫反应。有趣的是，我们的结果表明，通过基因疫苗接种，在各种受试小鼠品系中产生了不同的B细胞表位。
