周期型马来丝虫3-磷酸甘油醛脱氢酶基因真核表达载体的构建及DNA免疫研究

Construction of pcDNA3.1 -Brugia malayi glyceraldehyde phosphate dehydrogenase eukaryotic recombinant plasmid and study on its role in DNA immunity

目的构建周期型马来丝虫3-磷酸甘油醛脱氢酶（BmGAPDH）真核表达载体pcDNA3．1-BmGAPDH，并观察其在小鼠体内的细胞免疫应答效应。方法以周期型马来丝虫总RNA为模板，RT—PCR方法扩增目的基因片段。与pGEM—TEasy克隆载体连接，筛选出阳性克隆，经PCR和双酶切鉴定后，亚克隆至真核表达质粒pcDNA3．1，构建pcDNA3．1-BmGAPDH表达载体，将纯化的pcDNA3．1-BmGAPDH和CpG经后腿胫前肌免疫BALB／c小鼠，并设PBS对照组及空质粒对照组，共免疫3次，每次免疫间隔2周。用RTPCR方法检测肌肉组织内目的基因；用噻唑蓝（MTT）法、ELISA方法分别检测免疫小鼠T淋巴细胞刺激增殖指数和血清中细胞因子IFN-γ、IL-4水平。应用SPSS统计学软件进行样本均数的t检验。结果成功构建了pcDNA3．1-BmGAPDH真核表达载体，基因片段全长为1020bp，DNA序列分析与基因库已知基因序列同源性为99％。该真核表达载体免疫小鼠后，从小鼠肌肉组织扩增出目的基因重组质粒组小鼠淋巴细胞刺激增殖指数显著高于PBS对照组及空质粒对照组，淋巴细胞刺激增殖指数分别为1．398、1．006和1．017（P〈0．05）。重组质粒组IFN-γ、IL-4细胞因子水平均高于PBS对照组及空质粒对照组，分别为163．905、58．589、51．317ng／L和107．906、27．111、34．627ng／L（P〈0．05）。免疫佐剂CpG与疫苗同时注射可增强机体的免疫应答，表现为IFN-γ水平和免疫4周后淋巴细胞刺激增殖指数显著上升。结论pcDNA3．1Bm GAPDH真核表达载体能在小鼠体内表达并可诱导相关的细胞免疫应答。

Objective To construct the pcDNA3. 1-Brugia malayi （Bin） glyceraldehyde 3- phosphate dehydrogenase （GAPDH） eukaryotic recombinant plasmid and to study its effect on mouse cellular immunity response. Methods Total RNA was prepared from periodic Bin. The target gene fragments were amplified by reverse transcription-polymerase chain reaction （RT-PCR） technique and then were inserted into the cloning vector, pGEM-T Easy, and sub-cloned into pcDNA3.1. Purified pcDNA 3.1 BmGAPDH recombinant plasmid and CpG were injected into the anterior tibial muscle of BALB/c mice in order to induce host immunity response. Mice injected with PBS and mice injected with blank plasmid were prepared as controls. The mouse models were immunized for 3 times with an interval of 2 weeks. RT-PCR was utilized to detect target gene expression in the muscle tissue. MTT method was used to measure the immunized mice T lymphocytes stimulation index, while enzymelinked immunoassay （ELISA） was used to determine the serum interleukin （IL）-4 and interferon （IFN）-γ level. Means were compared using t test with SPSS software. Results The recombinant plasmid pcDNA3.1-BmGAPDH was constructed sueessfully. The target gene was 1020 hp long and its homology with known gene sequence in database was 99%. BmGAPDH gene in the injected muscle of the immunized mice was detected by PCR. The proliferation of spleen T lymphocytes was higher in peDNA3 BmGAPDH group than in the 2 control groups which were 1. 398, 1. 006 and 1. 017, respectively （P〈0.05）. The levels of IFN-γ and IL -4 in serums from the immunized mice were significantly higher than those of the PBS control group and blank plasmid control group which were 163. 905, 58. 589, 51. 317 and 107. 906, 27. 111, 34. 627, respectively （P〈0.05）. Immune adjuvant CpG could accelerate and boost antigen-specific immune responses induced by vaccine, which presented as significant increase of IFN -γ and lymphocyte proliferation at 4 weeks after immunization. Conclusion The recombinant eukaryotic plasmid pcDNA3. 1-BmGAPDH is constructed and could elicit cellular immune responses in immunized mice.