neuB1基因失活空肠弯曲菌突变株的构建及其临床意义

Construction and its clinical significance of the mutant Campylobacter jejuni O∶19 strain with inactivated neuB1 gene

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 the mutant of Campylobacter jejuni （Cj） O： 19 strain in lipopolysaccharide （LPS） and to identify the critical role at the terminal GM1-like structure containing SA residue of LPS in the pathogenesis of Cj-associated Guillain-Barre syndrome（GBS）. Methods DNA fragment of neuB1： ：Km^R was cloned into a suicide vector pGEM-T to construct a mutant vector pGEM-neuB1 ： ：Km^R identified by endonuclease digestion and PCR. The mutant vector was transformed into a GBS-associated O ： 19 Cj strain. PCR and RT-PCR were used for confirming the mutant strain. LPS of the mutant and wild strain of O： 19 Cj were analyzed by SDSPAGE and SA of the LPS was determined by periodate-resorcinol reaction and acidic ninhydrin reaction respectively. GM1-like epitope in the whole-cell lysates and LPS of the strains was detected by cholera toxin B subunit （ CTB）. Guinea pigs were immunized the purified wild and the mutant LPS respectively. Histological morphology of the teasing fibers of sciatic nerve after immunization was investigated. Results PCR product of 2259 bp was harvested and the DNA fragment neuB1 ： ： Kma was successfully cloned into the pGEM-T vector, which was identified by endonuclease digestions and PCR. Kma cassette was inserted into the neuB1 gene of Cj O： 19 strain. The mutant of Cj O： 19 strain deleting neuB1 gene was successfully constructed and absent transcriptional activity of neuB1 gene in the mutant strain was confirmed by PCR and RT-PCR. Good stability on keeping resistance to kanamycin and inactivity of neuB1 gene was still demonstrated. Structure of sialic acid was well demonstrated by both periodate-resorcinol reaction and acidic ninhydrin reaction in the LPS of wild strain but not in the mutant LPS; lysate and LPS of the wild strain could bind to CTB but not to those of the mutant. There was 17.3 % incidence of sciatic nerve fibers to be damaged after systemic immunization with wild LPS, but only 2.4% incidence in the mutant group. Conclusion A mutant of Cj O： 19 strain deleting neuB1 gene was successfilly constructed. There was no longer expression of SA component, which is the normal structure of LPS in wild strain. The feature of molecular mimicry between LPS of wild Cj strain and GM1 as well the capability of immunogenic damage to nerve were both lost in the mutant simultaneously. It is an evidence to the molecular mimicry hypothesis on the pathogenesis of Cj related GBS and SA epitope as the basic GMl-like structure in LPS.