中文摘要：

目的 制备人内皮高表达脂多糖相关因子 1（EOLA1）多克隆抗体,检测EOLA1在人脐静脉内皮细胞（HUVEG）中的表达.方法 复性成功表达EOLA1的蛋白样品（样品1和2）,二辛丁酸法检测2种蛋白样品浓度,应用肽质量指纹图谱（PMF）分析法对2种样品进行验证.采用与理论肽段符合率高的EOLA1蛋白样品免疫4只小鼠,同时取4只未免疫小鼠作对照.采集各小鼠血液分离血清,应用饱和硫酸铵盐析沉淀法纯化EOLA1多克隆抗体,ELISA法分析效价（数据以吸光度值表示）.蛋白质印迹法检测EOLA1在HUVEC中的表达.结果 蛋白样品1和2的浓度分别为0.124 16、0.132 15 mg/mL.PMF分析显示,样品1的理论肽段符合率为32%,样品2的理论肽段符合率为24%.选用蛋白样品1免疫小鼠,经检测抗体效价在1∶10 000以上;应用制备的EOLA1多克隆抗体可以检测到EOLA1蛋白在HUVEC中表达.结论 应用EOLA1原核表达蛋白免疫小鼠可以制备EOLA1多克隆抗体,并可用于检测EOLA1蛋白表达.

英文摘要：

Objective To prepare the polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1 （EOLA1） , and to determine the expression of EOLA1 in human umbilical vein endothelial cell （HUVEC）. Methods The protein samples （ sample 1 and 2） expressing EOLA1 were purified and renatured. The protein concentrations were determined with bicinchoninic acid assay. The protein samples were identified with peptide mass fingerprinting （PMF） analysis. Protein sample with higher coincidence rate of amino acid sequence with theoretic protein was used to inoculate 4 mice; another 4 mice inoculated with adjuvant were used as control. Serum was isolated from collected mice blood. Polyclonal antibody of EOLA1 was purified with saturated ammonium sulfate precipitation, and was determined with ELISA for the titer （ data were denoted by absorbance value）. The expression of EOLA1 in HUVEC was determined with Western blot. Results The concentration of protein sample 1 and 2 was respectively 0. 124 16 mg/mL and 0. 132 15 mg/mL. According to PMF analysis, the coincidence rate of amino acid sequence between protein samples and theoretic protein were 32% （ protein sample 1 ） and 24% （ protein sample 2）. The polyclonal antibody of EOLA1 with titer more than 1：10 000 was obtained from mice inoculated with protein sample 1. The expression of EOLA1 protein in HUVEC was determined with polyclonal antibody of EOLA1. Conclusions The polyclonal antibody of EOLA1 can be prepared by inoculating mice with EOLA1 prokaryotic expressing protein, which can be used for determination of EOLA1 protein.