CAJ | 학술논문

目的研究华北地区3株吉兰-巴雷综合征(Guillain-Barré syndrome,GBS)相关空肠弯曲菌(Campylobacter jejuni,C.jejuni)的cstⅡ基因序列并寻找GBS相关及GBS无关的两类空肠弯曲菌菌株间可能与GBS致病性相关的基因突变位点和蛋白高级结构变化,分析菌株间遗传进化关系.方法选取分离自GBS患者粪便并经动物模型证实为致GBS的3株空肠弯曲菌菌株进行培养并提取基因组DNA测序,与GenBank中GBS无关空肠弯曲菌基因序列进行配对对比,寻找两类菌株间可能导致空肠弯曲菌致病能力差异的cstⅡ基因碱基突变位点及蛋白高级结构改变,观察这种变化是否为GBS相关菌株的共性,并构建遗传进化树.结果与GBS无关菌株相比,3株致GBS菌株cstⅡ基因的氨基酸序列二级结构的第7个α螺旋的165～180区段均被打开形成了折叠或转角,该变化在其他GBS相关菌株的二级结构变化中同样得到体现.实验中75%的GBS相关菌株cstⅡ基因为双功能,25%GBS相关菌株及所有GBS无关菌株cstⅡ基因为单功能.LL株与实验中涉及的GBS相关菌株在进化上高度类聚.结论双功能cstⅡ可能与空肠弯曲菌的致GBS特性相关.LL株cstⅡ基因在一定程度上反映了亚洲地区的GBS相关菌株cstⅡ基因的序列特征,为进一步探索GBS发病的地域性特点并对GBS菌株进行监测提供了资料.
목적 연구화북지구3주길란-파뢰종합정(Guillain-Barré syndrome,GBS)상관공장만곡균(Campylobacter jejuni,C.jejuni)적cstⅡ기인서렬병심조GBS상관급GBS무관적량류공장만곡균균주간가능여GBS치병성상관적기인돌변위점화단백고급결구변화,분석균주간유전진화관계.방법 선취분리자GBS환자분편병경동물모형증실위치GBS적3주공장만곡균균주진행배양병제취기인조DNA측서,여GenBank중GBS무관공장만곡균기인서렬진행배대대비,심조량류균주간가능도치공장만곡균치병능력차이적cstⅡ기인감기돌변위점급단백고급결구개변,관찰저충변화시부위GBS상관균주적공성,병구건유전진화수.결과 여GBS무관균주상비,3주치GBS균주cstⅡ기인적안기산서렬이급결구적제7개α라선적165～180구단균피타개형성료절첩혹전각,해변화재기타GBS상관균주적이급결구변화중동양득도체현.실험중75%적GBS상관균주cstⅡ기인위쌍공능,25%GBS상관균주급소유GBS무관균주cstⅡ기인위단공능.LL주여실험중섭급적GBS상관균주재진화상고도류취.결론 쌍공능cstⅡ가능여공장만곡균적치GBS특성상관.LL주cstⅡ기인재일정정도상반영료아주지구적GBS상관균주cstⅡ기인적서렬특정,위진일보탐색GBS발병적지역성특점병대GBS균주진행감측제공료자료.
Objective To investigate the pathogenic mechanism of Campylobacter jejuni(C.jejuni) associated with Guillain-Barré syndrome(GBS) and provide strategy for gene modification, the cstⅡ gene from 8 GBS-associated C.jejuni strains were compared with that from 3 GBS-unrelated C.jejuni strains, getting the base and amino acid mutations, the changes of secondary structures and finding the region which may be responsible for the pathogenicity of C.jejuni inducing GBS. Methods Three GBS-associated C.jejuni strains isolated from stools of GBS patients in north China were selected and cultured, which has been confirmed as GBS-associated by animal model. After sequencing the genome of them, the nucleotide sequences of cstⅡ gene were got through sequence alignment. The nucleotide sequences and deduced amini acid sequences of 3 GBS-associated cstⅡ genes were compared with that from 3 GBS-unrelated C.jejuni strains through bioinformatics software, getting the base and amino acid mutations, the changes of secondary structures. Other 5 GBS-associated cstⅡ genes were also aligned to know whether the differences we got above makes sense. In this way the genetic differences between two kinds of C.jejuni strains may be found and speculating the gene region related to the pathogenicity of GBS became possible. Results The cstⅡgene of 3 GBS-associated C.jejuni strains were all composed of 876 base pairs. Compared with GBS-unrelated C.jejuni strains, there were 9 consistent mutation sites in cstⅡ gene of LL and QYT stains, leading to 3 consistent amino acid mutation. The amino acid mutation of 114 and 182 sites in LL and QYT stains existed in other 5 GBS-associated C.jejuni strains. The sole amino acid mutation of ZHX strain -169 site, located near the 182 site. The seventh α-helix(165-180 region)of the secondary structure of the amino acid sequence from GBS-associated strains were shorter than that from GBS-unrelated strains, and the shorter regions were opened to form β-sheet or coli, which also existed in other GBS-related strains in this study.75% of the GBS-associated cstⅡ genes were Asn-51, while 25% of the GBS-associated and all of the GBS-unrelated cstⅡ genes were Thr-51.LL strain showed highly identity to other GBS-unrelated strains in this study. Conclusion The 165-180 segment of secondary structures in cstⅡ gene from local 3 GBS-associated C.jejuni strains are probably the responsible region involved in inducing GBS. The senior structure changes in this region may affect the activity of sialyltransferase and the structures of ganglioside epitope, so that the C.jejuni can acquire the pathogenicity of GBS. This finding may give a clue to genetic modified site. The bi-functional cstⅡ of C.jejuni may be related to the pathogenicity of GBS. The cstⅡ of LL strain to some extent represents the characteristics of Asian strains, which may directs strains monitoring.