艰难梭菌鞭毛帽蛋白的功能研究  

Study on function of Clostridium difficile flagellar cap protein

作　　者：王德慧[1,2,3] 谭林林[3] 王琴[3] 陈芳红[3] 李涛[3] 王慧[3] 

机构地区：[1]安徽医科大学 [2]军事医学科学院微生物流行病研究所微生物组学与生物信息学研究室,合肥230032 [3]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《安徽医科大学学报》2014年第11期1582-1586,共5页Acta Universitatis Medicinalis Anhui

基　　金：北京自然科学基金(编号:7122134);国家重大科技专项重大新药创制(编号:2013ZX09304101)

摘　　要：目的验证艰难梭菌鞭毛帽蛋白FliD的黏附细胞活性以推测其在艰难梭菌感染中的作用,并鉴定其抗原性以测试其应用于候选艰难梭菌疫苗组分的可能性。方法 PCR获取VPI 10463全长fliD基因,构建pET-22b-fliD原核表达质粒,转化至BL21（DE3）中,诱导表达,Ni2＋亲和层析纯化,进行N端测序验证。蛋白3次皮下免疫大耳白兔后间接ELISA法检测其血清特异IgG水平并鉴定抗原性。间接免疫荧光染色后流式细胞术检测黏附Vero细胞能力。结果 VPI10463的fliD基因钓取成功,与GenBank公布的其他9株艰难梭菌的fliD基因相似度在89%～100%。pET-22b-fliD表达质粒构建成功,表达蛋白大小为56 ku,N端测序序列为MSSIS,与预期一致。一步纯化得到的蛋白纯度为99%以上。ELISA检测3次免疫后效价达到105。用流式细胞术检测其可以黏附在Vero细胞表面。结论构建的pET-22bfliD表达质粒成功表达FliD,具有黏附细胞活性,在艰难梭菌感染过程中起黏附因子作用,其具有较高的抗原性可以作为预防艰难梭菌感染的候选抗原之一。Objective To identify the adherence capability and antigenicity of Clostridium difficile （ C. difficile ） flagellar cap protein FliD and to investigate its role in C difficile infection and its potentiality to be a candidate vac- cine component. Methods The full-lengthfliD was amplified by PCR from C. difficile strain VPI 10463. The prod- uct was cloned into pET-22b, and the recombinant plasmid was transformed into E. coil BI21 （DE3） strains, posi- tive clones were induced by isopropyl-l-thio-β-galactopyranoside （IPTG） to express. Recombinant protein with His- tag its was purified using Ni^2＋ NTA Sephrose and confirmed by N-terminal sequencing. Enzyme linked immunosor- bent assay （ELISA） was applied to detect specific IgG against FliD in serum of a rabbit which was immunized by three subcutaneous injections. Vero incubated with FliD were detected in a Flow Cytometer （FCM） after indirect immunofluorescence stain to check out adhesion of FliD. Results The complete fliD gene was amplified from strain VPI10463 with sequence of 89%～ 100% identity betweennine other C. difficile strains published in GenBank. The pET-22b-fliD recombinant plasmid expressed successfully a protein of 56 ku with the N-terminal sequence MSSIS which exhibited up to 100% identity as predicted. FliD with purity of 99% was obtained by one-step expression. The rabbit developed a titer of 105 prominent anti-FliD immunoglobulin G in serum after three immunization. FCM provided the proof for FliD＇s function as a cell surface-adherence factor. Conclusion The recombinant protein FliD of VPI10463 has been expressed by pET-22b-fliD plasmid in E. coil. The phenomenon that FliD can adhere to Vero in vitro confirms its role as an adherence factor in C. difficile infection. FliD is high antigenic, in addition, which makes it one of the possible candidate antigen in preventing C. difficile infection.

关 键 词：艰难梭菌 鞭毛帽蛋白FliD 重组表达 流式细胞 术 黏附因子 

分 类 号：R378.8[医药卫生—病原生物学] Q812[医药卫生—基础医学]