抗Ⅱ型登革病毒蛋白抗体的制备和特点分析  

Preparation and characteristic analysis of anti-dengue virus serotype 2 antibodies

作　　者：魏建春[1] 崔晓云[1] 吴娜[1] 安静[1] 

机构地区：[1]首都医科大学基础医学院病原生物学系微生物学教研室,北京100069

出　　处：《微生物与感染》2015年第3期147-153,共7页Journal of Microbes and Infections

基　　金：国家自然科学基金(81301435;81401676;81471957)

摘　　要：本研究旨在制备和鉴定小鼠抗Ⅱ型登革病毒（DENV‐2）10种蛋白的抗体，为后续相关研究提供实验材料。利用真核表达载体pReceiver构建DENV‐210种蛋白的重组质粒，提取质粒 DNA ，肌内注射免疫小鼠，共免疫4次。末次免疫后2周取小鼠血清，利用DENV‐2感染的Vero细胞和DENV‐2各蛋白的稳定表达细胞，通过酶联免疫吸附试验（ELISA）、间接免疫荧光法（IFA）和蛋白免疫印迹法评价免疫效果，分析抗体的特点。DNA免疫小鼠后获得抗DENV‐210种蛋白的抗血清，抗体效价波动于1∶400～1∶16127之间，以抗E蛋白抗体效价最高，达1∶16127，而抗NS3、NS4b、NS5蛋白抗体效价较低，仅为1∶400。利用DENV‐2感染的Vero细胞和稳定表达病毒蛋白的EAhy926细胞进行IFA染色，抗DENV‐2各蛋白的抗血清均可特异性识别DENV‐2抗原。蛋白免疫印迹结果显示，抗E、NS1、NS4b和NS5蛋白抗体能识别热变性蛋白，其他抗血清未呈现阳性反应条带。本研究提示，DNA免疫小鼠所获得的抗DENV‐2各蛋白抗体能特异性识别自然感染或模拟自然感染状态下的DENV‐2蛋白，可为后续相关研究提供工具，也表明DNA免疫法可作为抗体制备的一种策略。Eukaryotic expression vector containing different proteins （C ,M ,E and NS1‐5） of dengue virus serotype 2 （DENV‐2 ） were constructed and intramuscularly injected directly to mice for developing antibodies against DENV‐2 .Two weeks after the fourth immunization ,sera were collected and the immune effects were evaluated by enzyme‐linked immunosorbent assay （ELISA） ,indirect immunofluorescence assay （IFA） and Western blotting .Specific antibodies could be obtained by DNA immunization ,and the titers of these antibodies fluctuated between 1∶400‐1∶16 127 .Of those ,the antibody against protein E showed the highest titer at 1∶16 127 whereas the antibodies against proteins NS3 ,NS4b and NS5 had the lowest titer at 1∶ 400 . The results showed that all antibodies acquired by DNA immunization could produce specific immunofluorescent reactions when tested using DENV‐2‐infected Vero cells , suggesting that antibodies prepared by DNA immunization could specifically identify naturally infected DENV‐2 .Antibodies against proteins E ,NS1 ,NS4b and NS5 could also identify heat‐denatured proteins .The results generated from this study provided the base for further research .

关 键 词：登革病毒 结构蛋白 非结构蛋白 抗体 EAhy926细胞 

分 类 号：R392[医药卫生—免疫学]