结核分枝杆菌热休克蛋白16．3及其合成肽诱导小鼠免疫应答的研究  

The immunological responses induced by Mycobacterium tuberculosis heat shock protein 16. 3 and its synthetic peptide in mice

作　　者：师长宏[1] 张廷芬[1] 朱德生[1] 张海[1] 白冰[1] 赵勇[1] 岳晨莉[2] 赵雷[2] 刘建利[1] 

机构地区：[1]第四军医大学实验动物中心,西安710032 [2]西京医院呼吸内科

出　　处：《中华结核和呼吸杂志》2009年第8期603-607,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家高技术研究发展（863）计划资助项目（2007AA022473）;国家自然科学基金资助项目（30670116）;陕西省自然科学基金资助项目（2006C218）

摘　　要：目的比较MTB的热休克蛋白（HSP）16．3和HSP16．3合成肽在小鼠体内诱导的免疫应答及保护力。方法80只BALB／c小鼠分为5组，3个实验组：蛋白组：HSP16．3／溴化二甲基双十八烷酸铵（DDA）／单磷酰脂质A（MPL），合成肽组：HSP16．3合成肽／DDA／MPL，佐剂组：HSP16．3合成肽／弗氏不完全佐剂组；2个对照组：卡介苗组和生理盐水组。每只小鼠蛋白和合成肽免疫剂量为50μg／次，共免疫3次，间隔2周。卡介苗为单次免疫5×10^6CFU／只。分别于第1次免疫后0、2、4、6、8周采集血液，酶联免疫吸附试验（ELISA）法检测血清中抗HSP16．3的抗体浓度及第8周时血清中抗体滴度。末次免疫完成后4周，每组取8只小鼠分离脾淋巴细胞，用HSP16．3刺激后，噻唑蓝法测定脾淋巴细胞的增殖指数，夹心ELISA法检测相同抗原刺激下脾淋巴细胞诱生的γ-干扰素水平。每组剩余小鼠用10’CFU的H37Rv毒株攻击，4周后取小鼠脾脏和肺脏，组织匀浆，倍比稀释后涂7H10平板，4周后计数菌落数。采用LSD-t检验统计学分析。结果抗HSP16．3抗体在免疫的前4周均增长迅速，之后趋于缓和。免疫8周时，3个实验组（蛋白组、合成肽组和佐剂组）抗体水平分别为1：2000、1：2000和1：1000，均高于卡介苗组（1：500）。3个实验组（蛋白组、合成肽组和佐剂组）的脾淋巴细胞增殖指数（3．13±0．18、3．21±0．21和2．40±0．15）均显著高于卡介苗组（1．67±0．12）和生理盐水组（1．04±0．09），蛋白组和合成肽组均显著高于佐剂组。3个实验组诱生的γ-干扰素水平·（182±6）、（194±9）和（179±8）mg／L]均显著低于卡介苗组[（275±10）mg／L]，高于生理盐水组[（71±3）mg/L]，3个实验组之间均无显著差别。3个实验组均对MTB在脾脏[细菌数为（6．74±0．14）-（6．81±0．28）lgCFU]和肺脏[细菌数为（5．74±0．27）-（6．65Objective To evaluate the immune responses and resistance against Mycobacterium tuberculosis（MTB） infection in the mice induced by HSP16. 3 of MTB and its synthetic peptide. Methods BALB/c mice were immunized subcutaneously 3 times at 2 week interval at the base of tail. The doses of HSP16. 3 protein and synthetic peptide were both 50 μg each time. A single dose of BCG （5 × 106 CFU/ mouse） was used to immunize the mice. The concentrations of specific antibodies in serum obtained at 0, 2, 4, 6, 8 weeks after the first immunization and the titer of serum obtained at 8th week, were analyzed by enzyme linked immunosorbent assay （ELISA）. Four weeks after the final immunization, 8 mice from each group were sacrificed and single-cell suspensions of splenocytes were prepared, some of which were used for lymphocyte proliferation by MTT colorimetry with HSP16. 5 stimulation, and the remaining cells were used for IFN-γ level assay by sandwich ELISA. The remaining mice in each group were challenged intravenously with 105 colony forming units （CFU） of MTB H37 Rv and were sacrificed 4 weeks after infection, and the number of bacteria in the spleens and lungs were determined by plating serial dilutions of homogenized tissue on Middlebrook 7H10 agar. The statistical significance of differences among means was assessed by an LSD-ttest. Results The level of specific antibody to HSPI6. 3 protein and the peptide increased rapidly in the former ,1 weeks and moderately in the later weeks. The average antibody-specific titers of 3 experiment groups （ HSP16. 3 protein ＋ DDA ＋ MPL, synthetic peptide ＋ DDA ＋ MPL and synthetic peptide ＋ IFA） were higher than the BCG group. The indexes of spleen lymphocyte proliferation （SI） of the 3 experiment groups （3.13 ±0. 18, 3.21 ±0. 21 and 2. 40±0. 15） were significantly higher than the BCG group （ 1.67 ±0. 12） and the saline group （ 1.04±0. 09） respectively. The SI of HSP16. 3 protein ＋ DDA ＋ MPL group （3.13 ±0. 18） and synthetic peptid

关 键 词：分枝杆菌 结核 热休克蛋白质类 疫苗 亚单位 感染 

分 类 号：R686[医药卫生—骨科学]