布鲁杆菌侵染巨噬细胞过程中与外膜蛋白25作用的受体筛选  

Screening of receptor that interacted with outer membrane protein 25 during macrophage infection by Brucella abortus

作　　者：李亚[1] 王远志[2] 陈创夫[3] 

机构地区：[1]新疆石河子大学生命科学学院,832000 [2]新疆石河子大学医学院,832000 [3]新疆石河子大学动物科技学院,832000

出　　处：《中华地方病学杂志》2014年第6期596-600,共5页Chinese Journal of Endemiology

基　　金：国家973项目（2010CB530203）;国家科技支撑计划（2013BAI05B05）;国际科技合作项目（2013DFA32380）

摘　　要：目的 筛选布鲁杆菌侵染巨噬细胞过程中与布鲁杆菌外膜蛋白（OMP）25作用的受体.方法 利用已经构建的pET32a-omp25表达载体,在异丙基硫代半乳糖苷OPTG）诱导下表达重组OMP25.蛋白,Ni-NTA柱纯化OMP25蛋白;采用噬菌体展示技术从M13噬菌体环形多肽展示库中进行环形7肽（c7c）的筛选;生物信息学软件分析筛选出潜在多肽,用酶联免疫吸附试验（ELISA）验证OMP25与多肽分子的结合活性;合成结合力高的7肽分子,用菌落形成单位计数和ELISA法检测其在布鲁杆菌感染小鼠巨噬细胞过程中的功能.结果 噬菌体展示技术共筛选出42个7肽分子;生物信息学软件分析筛选出4个潜在多肽,ELISA证实4个7肽分子（ST-7、MT-7、TT-7和SN-7）能够与布鲁杆菌OMP25蛋白具有高亲和力;菌落形成单位计数结果显示：多肽分子ST-7、MT-7、TF-7和SN-7在布鲁杆菌黏附或抑制胞内寄生中均有一定作用.ST-7、MT-7、TF-7和SN-7对牛种布鲁杆菌2308菌株的抑制率范围分别为14.4％～51.8％、49.6％～69.5％、43.2％～74.4％、57.5％ ～82.2％.ELISA结果显示,与对照组比较,ST-7和MT-7多肽实验组肿瘤坏死因子（TNF-α）的量在各个时间点的差异无统计学意义（P均＞ 0.05）;而TF-7和SN-7多肽实验组TNF-α的量在8h前差异无统计学意义（P均＞0.05）,但在12、24h时促进TNF-α的分泌,差异具有统计学意义（P均＜0.05）.结论 筛选获得4个有效的OMP25作用受体,其中TF-7、SN-7对布鲁杆菌2308侵染小鼠巨噬细胞既抑制入侵又抑制胞内生存,ST-7、MT-7对其只起抑制入侵的作用.Objective Receptor was screened to find out the interaction between outer membrane protein 25 of Brucella（OMP25） and Brucella infected macrophages.Methods Recombinant OMP25 protein was expressed and purified.The 7 peptide was screened out with OMP25 protein by phage display technology.OMP25-7 peptide interactions were further confirmed by enzyme linked immunosorbent assay（ELISA） after candidate peptides were predicted with bioinformatics software.Four potential 7 peptides were synthetized.Their functions were estimated after Brucella abortus 2308 infected murine macrophages RAW264.7.Results Forty-two cyclic 7 peptides were screened out based on purified OMP25 by the M13 Phage Library Kit.Four peptides,named as ST-7,MT-7,TF-7 and SN-7,were confirmed with ELISA.Colony forming unit（CFU） result demonstrated ST-7,MT-7,TF-7 and SN-7 showed some function for inhibition of intracellular parasite.ST-7 inhibition ratio was 14.4%-51.8% for Brucella abortus 2308; MT-7 inhibition ratio was 49.6%-69.5% for Brucella abortus 2308; TF-7 inhibition ratio was 43.2%-74.4% for Brucella abortus 2308; SN-7 inhibition ratio was 57.5%-82.2% for 2308.ELISA result showed,that compared to control group,tumor necrosis factor α（TNF-α）increased no statistical significance in each time point of ST-7,MT-7 experiment groups （all P 〉 0.05）.There was no statistical significance in 8 h ago of TF-7,SN-7 experiment groups （all P 〉 0.05）,but there was statistical significance in 12,24 h （all P 〈 0.05）.Conclusions We have obtained four effective OMP25 receptors through phage display technology; TF-7 and SN-7 have inhibited Brucella abortus 2308 invasion and intracellular survival in murine macrophage cells,while ST-7 and MT-7 have only prevented the invasion against Brucella abortus 2308.

关 键 词：布鲁杆菌 外膜蛋白25 噬菌体展示技术 巨噬细胞 outer MEMBRANE PROTEIN 25 

分 类 号：R516.7[医药卫生—内科学]