论文部分内容阅读
目的构建唾液酸合成酶基因1(neuB1)失活、脂多糖(LPS)中唾液酸(SA)缺失的空肠弯曲菌(Campylobacterjejuni,Cj)O∶19突变株,以确认CjLPS中,含SA基团的终端GM1样结构在Cj相关格林-巴利综合征(GBS)发病机理中的关键作用。方法从已有neuB1基因失活的Cj肠炎相关菌株O∶2DNA中,将含卡那霉素抗性基因(KmRcassette)的neuB1突变片段———neuB1::KmR克隆到pGEM-T载体,形成突变载体pGEM-neuB1::KmR,进一步转化到与GBS相关的CjO∶19野生株,获得CjO∶19突变株,行PCR和RT-PCR鉴定。SDS-PAGE分析CjLPS,过碘酸-间苯二酚和酸性茚三酮反应法分别检测CjLPS中SA。霍乱毒素B亚单位(CTB)结合反应检测Cj菌体裂解物及LPS中神经节苷脂GM1模拟结构。最后以突变株及其同源野生株LPS为抗原分别免疫豚鼠,第5周取坐骨神经原纤维分离。结果neuB1基因突变片段———neuB1::KmR成功克隆到pGEM-T载体中,进而构建得neuB1基因失活的Cj突变株。后者LPS已丧失SA基团且不能与CTB结合,表明其菌体裂解物和LPS中已不存在GM1模拟结构。野生株LPS免疫组中,17.3%的坐骨神经原纤维发生免疫性损伤,明显高于PBS对照组(1.6%)和突变株LPS免疫组(2.4%);而突变株LPS免疫组与PBS对照组间差异无统计学意义。结论成功构建neuB1基因失活的CjO∶19突变株,其LPS已缺失野生株所具有的SA基团及与GM1的分子模拟特性,且不能再诱导活体动物的周围神经免疫性损伤,有力证实了关于CjLPS诱发GBS的分子模拟推论,SA是该交叉免疫反应中抗原的重要成分。
OBJECTIVE: To construct a mutated strain of Campylobacterjejuni (Cj) O:19 with inactivated sialic acid synthetase gene 1 (neuB1) and sialic acid (SA) in lipopolysaccharide (LPS) Terminal GM1-like structure in the pathogenesis of Cj-related Guillain-Barre syndrome (GBS). Methods The neuB1 mutant containing the kanamycin resistance gene (KmRcassette) --- neuB1 :: KmR was cloned into pGEM-T vector from the O: 2 DNA of Cj colitis associated with inactivation of neuB1 gene to form a mutation The vector pGEM-neuB1 :: KmR was further transformed into CjO: 19 wild-type strain related to GBS to obtain CjO:19 mutant strain, which was identified by PCR and RT-PCR. SDS-PAGE analysis of CjLPS, periodic acid - resorcinol and acid ninhydrin reaction were detected in SA CjLPS. Cholera toxin B subunit (CTB) binding reaction detected Cj bacterial lysate and ganglioside GM1 simulated LPS structure. Finally, the mutant strain and its homologous wild-type LPS antigen were immunized guinea pigs, the first 5 weeks of sciatic nerve fibrillation. Results neuB1 gene mutation fragment --- neuB1 :: KmR successfully cloned into pGEM-T vector, and then constructed neuB1 gene inactivated Cj mutant. The latter LPS had lost the SA group and could not bind to CTB, indicating that the GM1 mimicry was absent in both bacterial lysates and LPS. In wild-type LPS group, 17.3% of sciatic nerve fibroblasts were immunocompromised, which was significantly higher than that of PBS control group (1.6%) and LPS-immunized group (2.4%), while the mutant LPS immunized group and PBS control group The difference was not statistically significant. Conclusions The CJO: 19 mutant with neuB1 gene inactivation was successfully constructed. The LPS-deficient SA group and the molecular mimicry of GM1 were not found in LPS, and the peripheral nerve immunological damage of living animals could not be induced any more Molecular modeling of CjLPS-induced GBS Corollary, SA is an important component of antigens in this cross-immune response.