CAJ | 학술논문

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。
본연구지재제비화감정소서항Ⅱ형등혁병독（DENV‐2）10충단백적항체，위후속상관연구제공실험재료。이용진핵표체재체pReceiver구건DENV‐210충단백적중조질립，제취질립 DNA ，기내주사면역소서，공면역4차。말차면역후2주취소서혈청，이용DENV‐2감염적Vero세포화DENV‐2각단백적은정표체세포，통과매련면역흡부시험（ELISA）、간접면역형광법（IFA）화단백면역인적법평개면역효과，분석항체적특점。DNA면역소서후획득항DENV‐210충단백적항혈청，항체효개파동우1∶400～1∶16127지간，이항E단백항체효개최고，체1∶16127，이항NS3、NS4b、NS5단백항체효개교저，부위1∶400。이용DENV‐2감염적Vero세포화은정표체병독단백적EAhy926세포진행IFA염색，항DENV‐2각단백적항혈청균가특이성식별DENV‐2항원。단백면역인적결과현시，항E、NS1、NS4b화NS5단백항체능식별열변성단백，기타항혈청미정현양성반응조대。본연구제시，DNA면역소서소획득적항DENV‐2각단백항체능특이성식별자연감염혹모의자연감염상태하적DENV‐2단백，가위후속상관연구제공공구，야표명DNA면역법가작위항체제비적일충책략。
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 .