肺炎支原体P1蛋白片段免疫学活性及黏附功能的研究  

Characterization of immunologic and adhesive abilities of Mycoplasma pneumoniae P1 protein seg- ment

作　　者：朱翠明[1,2] 汪世平[1] 吴移谋[2] 高顺利[3] 余敏君[2] 陈曦[4] 

机构地区：[1]中南大学湘雅医学院病原生物学系,长沙410078 [2]南华大学医学院微生物学与免疫学教研室 [3]南华大学第一附属医院儿科 [4]湖南省疾病预防控制中心

出　　处：《中华微生物学和免疫学杂志》2012年第8期706-710,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81072418）;湖南省教育厅优秀青年基金（098088）;湖南省高校科技创新团队资助项目（湘教通[2010]212号）

摘　　要：目的了解肺炎支原体（Mycoplasma pneumoniace，Mp）P1蛋白第1125～1395氨基酸片段（P1C蛋白）的免疫学活性及其细胞黏附作用。方法构建用于表达重组P1C片段（rP1C）的原核表达载体pGEX6p-2／p1c，采用SDS-PAGE和Westernblot鉴定rP1C。采用基于GST的亲和层析法提纯rPIC，提纯的rPC免疫BALB／c小鼠，ELISA检测小鼠抗rP1C血清的效价。采用Westernblot检测rPIC对Mp感染患者血清的免疫反应性。采用间接免疫荧光法检测rP1C黏附HeLa细胞及其免疫血清黏附抑制作用。结果所构建的原核表达系统能有效表达相对分子质量约为66×10^3的可溶性rP1C。rP1C免疫小鼠后，其抗血清ELISA效价高达1：64000。rPIC能被Mp感染者血清及小鼠抗rPIC血清识别并与之结合。rP1C能黏附HeLa细胞，其抗血清可阻断Mp对HeLa细胞的黏附，该黏附阻断作用随抗血清浓度增高而增强。结论rP1C具有良好的免疫原性和免疫反应性及黏附细胞功能，可作为Mp疫苗及血清学检测的候选抗原。Objective To deiermine the immunogenic and adhesive abilities of a segment （ P1C protein ） that located at the carboxy terminal region ofP 1 protein （ 1125 to 1395 amino acids ） . Methods A recombinant prokaryotic vector （pGEX6p-2/plc） was constructed for P1C protein expression in E. coli BL21DE3. The expressed target recombinant protein （rP1C） was identified using SDS-PAGE and Western blot assay, and then extracted by GST-based affinity chromatography. The purified rP1 C was used to immu nize BALB/c mice to obtain rP1 C-antiserum and titer of the antiserum was determined by ELISA. Immunoreactivity of the rP1C to the sera form M. pneumoniae-infected patients was detected using Western blot assay, while activity of the rP1C adhering to HeLa cells as well as adhesion blockage of the rP1C antiserum were de- tected using indirect immunofluorescence assays. Results The constructed prokaryotic expression system could efficiently express soluble rP1C with a relative molecular weight of 66×103. The antiserum from rP1 C- immunized mice showed an ELISA titer as high as 1：64 000. Both the M. pneumoniae-infected patients＇ sera and the mouse antiserum against rP1C could recognize as well as combine with the rP1C. rP1C could adhere to HeLa cells and the adhesion could be blocked by the mouse antiserum with an antiserum concentration-de- pendent manner. Conclusion P1C, a segment of M. pneumoniae P1 protein, possesses powerful immuno- genicity and immunoreactivity and cell-adhered activity, indicating the protein segment can be used as an an- tigen candidate for developing vaccines and serological diagnostic methods of M. pneumoniae-induced diseases.

关 键 词：肺炎支原体 P1蛋白片段 免疫学活性 黏附 

分 类 号：R563.1[医药卫生—呼吸系统]