抗Toll样受体4胞外C端结构域单克隆抗体制备及其治疗脓毒症的初步效果  

Preparation of anti-TLR4 extracellular C-terminal domain monoclonal antibody and its application to sepsis

作　　者：陈锋[1] 刘英富[2] 李光宗[3] 张益[3] 郁硕 侯世科[1] 

机构地区：[1]天津医科大学研究生院,300070 [2]武警后勤学院附属医院灾害应急救援医学全军重点实验室 [3]武警后勤学院附属医院救援医学研究所

出　　处：《中华创伤杂志》2016年第7期655-660,共6页Chinese Journal of Trauma

基　　金：基金项目：天津市科技计划（14ZCDZSY00033）;天津市应用基础与前沿技术研究计划（14JCQNJC12600）;全军重点实验室开放基金（JY1402）;武警后勤学院附属医院种子基金（FYM201514）

摘　　要：目的制备针对Toll样受体4（TLR4）胞外C端结构域的单克隆抗体，并探讨抗TLR4C端单克隆抗体对脓毒症的影响。方法原核表达、丙烯葡聚糖凝胶（SephacrylS-100）纯化获得大鼠来源的TLR4C端多肽（氨基酸序列：368—579，命名为TLR4-C），免疫6～8周雌性Balb／c小鼠，经细胞融合、抗体筛选、纯化等程序制备抗TLR4-C单克隆抗体，采用Western印迹、细胞免疫荧光对制备的TLR4-C单克隆抗体进行特异性检测。通过细胞实验和脓毒症动物模型实验进行抗体活性测定。（1）细胞实验：100μg/ml抗体加入培养的NR8383作用1h，加入10ng／ml脂多糖（LPS）继续培养12h后，ELISA检测培养基中肿瘤坏死因子-α（TNF-α）的释放水平；（2）脓毒症动物模型实验：40只SD大鼠按随机数字表法分为对照抗体组、B—D5抗体组，每组尾静脉分别注射50mg／kg相应抗体，1h后经腹腔给予10mg／kg致脓毒症剂量的LPS，4h后检测两组血清TNF—α含量，并连续72h观察两组动物存活情况。结果获得3株特异性高、且能结合TLR4-C和TLR4全蛋白的抗TLR4-C单克隆抗体。细胞活性测定显示，1株单克隆抗体（B—D5）能明显抑制细胞TNF—α的释放，其余2株对抑制细胞TNF-α的作用较弱。动物模型实验显示，B—D5抗体组TNF-α水平为（1．54±0．18）ng／ml，明显低于对照抗体组的（0．51±0．10）ng／ml（P〈0．01），B—D5抗体组存活率（70％）明显高于对照抗体组（30％）（P〈0．05）。结论制备的抗TLR4C端单克隆抗体具有很好的TLR4中和作用，明显提高大鼠的存活率，对LPS诱发的脓毒症具有较好的保护作用。Objective To prepare the investigate its effect in treatment of sepsis. anti-TLR4 C-terminal domain monoclonal antibody and Methods TLR4 C-terminal polypeptide （amino acid sequence： 368-579, named as TLR4-C） was obtained through prokaryotic expression and Sephacryl S-100 gel purification, and then was used to immunize female Balb/c mice （6-8 weeks old）. After cell fusion, antibody screening and purification, monoclonal antibody specific for the C terminal of TLR4 was obtained. Specificity of monoclonal antibody was detected by Western blot and cell immunofluorescence. In vitro antibody activity test, NR8383 was cultured for 1 h with adding antibody （100 μg/ml） and then 12 h after adding lipopolysaccharide （LPS） （10 ng/ml）, and level of tumor growth factor （TNF）-α in the culture medium was tested by ELISA. In vivo septic animal experiment, 40 SD rats were assigned to control antibody group （ n = 20） and anti-TLR4 monoclonal antibody group （ n = 20） according to therandom number table. Each group was rejected 50 mg/kg corresponding antibodies via caudal vein for 1 h, and then LPS （ 10 mg/kg） via intraperitoneal injection for 4 h. Blood samples from caudal vein of ten rats in each group were collect to test the serum level of TNF-α . The rest rats in each group were used to measure the animal survival rate within 72 h. Results Three highly specific anti-TLR4 monoclonal antibodies were obtained and could combined with TLR4-C and TLR4 holoprotein. In vitro cell activity study indicated only one monoclonal antibody could obviously inhibit the release of TNF-α. In vivo animal experiment showed serum TNF-α level in anti-TLR4-C antibody group was （1.54 ± 0.18 ） ng/ml, significantly lower than （0.51 ±0.10） ng/ml in antibody control group （ P 〈 0.01 ）. Animal survival rate in anti-TLR4-C antibody group was 70% , higher than 30% in antibody control group （P 〈 0.05 ）. Conclusion Anti-TLR4-C monoclonal antibodies have great capacity to neutralize TLR4 and good pr

关 键 词：TOLL样受体4 抗体 单克隆 脓毒症 

分 类 号：R459.7[医药卫生—急诊医学]