弓形虫棒状体蛋白2和膜表面蛋白1复合抗原优势表位免疫原性研究  

lmmunogenicity of dominant epitopes of complex antigen rhoptry protein 2-sunface protein 1 derived from Toxoplasma gondii

作　　者：吕金辉[1] 汪文寰 熊一融 杜王琪 叶璐璐[1] 张丽芳[1] 李文姝[1] 

机构地区：[1]温州医科大学微生物学与免疫学教研室,325032

出　　处：《中华传染病杂志》2016年第1期32-38,共7页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金（81371668）

摘　　要：目的分析弓形虫棒状体蛋白2（ROP2）和膜表面蛋白1（SAGl）复合抗原免疫优势表位的免疫原性。方法利用生物信息学技术预测和筛选出弓形虫复合抗原ROP2-SAG1免疫优势T、B淋巴细胞表位组合，将优势表位相应基因片段克隆至pET32a（＋）载体并转化至表达感受态（大肠埃希菌Rosetta菌株）中诱导表达，通过离子螯合亲和层析柱（Ni—NTA）纯化表达产物，经SDS—PAGE鉴定表达产物纯化效果。以纯化的优势表位蛋白作为免疫原，皮下多点注射日本大耳兔，设pET32a（＋）载体蛋白和PBS为对照，以纯化的优势表位蛋白作包被抗原，ELISA法检测第0、2和4周免疫兔血清中表位特异性抗体IgG的产生时间及效价，免疫斑点实验验证免疫兔血清多克隆抗体的特异性。以纯化的优势表位免疫BALB／c小鼠，酶联免疫斑点分析技术检测免疫鼠脾细胞7干扰素产生能力。结果在原核表达体系中获得了相对分子质量为30000的优势表位融合表达产物。纯化的表位蛋白免疫大耳白兔血清中可检测到相应的表位特异性抗体IgG，其抗体水平随着免疫次数的增加而呈现逐渐升高的趋势，优势表位免疫组第2、4周兔血清抗体显著高于载体pET32a（＋）蛋白对照组（1．454±0．098比0．616±0．084，F=0．000，P〈0．05；2．299±0．224比1．580±0．192，F=0．112，P〈O．05），第4周的免疫血清多克隆抗体效价可达1：40960。免疫斑点实验证实多克隆抗体针对优势表位具有良好的特异性。原核体系制备的优势表位蛋白免疫鼠脾细胞受3种不同的CTL表位肽刺激产生的7干扰素水平，分别为表位肽1[（19．333±1．528）／10万细胞]、表位肽2[（40．333±1．528）／10万细胞]、表位肽3E（70．667±1．890）／10万细胞]，与PBS免疫对照组[表位肽1（1．033±0．150）／10万细胞、表位肽2（1．045±0．110）／10万细胞、表位肽3（1．041±Objective To analyze the immunogenicity of dominant epitope of complex antigen of rhoptry protein 2 （ROP2） and major surface protein 1 （SAG1） derived from Toxoplasrna gondii （T. gondii）. Methods Dominant epitope of ROP2-SAG1 containing both dominant T- and B-cell epitopes was predicted and selected from TI gondii with bioinformatics methods. The gene fragment cloned into pET32a expression vector was transformed into the competent cell （Escherichia coli strain Rosetta） and expressed under the induction. The protein purified by nitrilotriacetic acid （Ni-NTA） agarose resin were finally identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Japanese rabbits were immunized subcutaneously with purified epitope protein in contrast with control group immunized with pET32a protein or phosphate buffevred saline （PBS）. The sera from immunized rabbits were collected at week 0, week 2 and week 4 for determination of epitope-specific antibody IgG using enzyme-linked immunosorbent assay, and immunodot assay was used to further confirm the specificity of antibody. After BALB/c mice were immunized with purified epitope proteins, the capacity of production of interferon-γ （IFN-γ） in splenocytes was detected by enzyme-linked immunospot assay. Results Relative molecular weight 30 000 of dominant epitope was derived from prokaryotic system. Then the rabbits immunized with purified dominant epitope could produce corresponding epitope-specific antibody IgG. And with the increased frequency of immunization, the level of antibody gradually increased. At week 2 and 4, higher antibody response were observed in group of rabbit immunized with dominant epitope than those of control group （1.454±0.098 vs 0. 616±0. 084, F=0. 000, P〈0. 05; 2.299±0.224 vs 1. 580±0. 192, F= 0. 112, P〈0. 05）. The antibody titer at week 4 was as high as 1：40 960. Immunodot assay further confirmed the antibody specificity against the dominant epitope. The level of IFN-7 in splenocytes from mice immunized w

关 键 词：弓形虫 ROP2-SAG1 表位 免疫原性 

分 类 号：R392[医药卫生—免疫学]