周期型马来丝虫半胱氨酸蛋白酶抑制剂和3-磷酸甘油醛脱氢酶复合基因真核表达载体的构建及免疫研究  

Construction of periodic Brugia malayi cysteine protease inhibitor and glyceraldehydes-3-phosphate dehydrogenase eukaryotic recombinant plasmid and its immunogenicity

作　　者：陆施娟[1] 方政[1] 张赛楠[1] 王慧[1] 方浩[1] 童海燕[1] 徐邦生[1] 

机构地区：[1]南通大学医学院寄生虫学教研室,226001

出　　处：《中华传染病杂志》2012年第5期259-263,共5页Chinese Journal of Infectious Diseases

基　　金：基金项目：江苏省高校研究生科研创新计划资助项目（2011653）;南通市应用研究计划资助项目（K2009027）

摘　　要：目的构建周期型马来丝虫（Bm）半胱氨酸蛋白酶抑制剂（CPI）和3-磷酸甘油醛脱氢酶（GAPDH）重组复合基因真核表达质粒，观察其在小鼠体内的细胞免疫应答效应。方法将重组质粒pGEM—T／BmCPI和pGEM—T／BmGAPDH以及真核重组表达载体分别进行双酶切，构建真核重组表达质粒pcDNA3．1（＋）-BmCPI／BmGAPDH。将纯化的pcDNA3．1（＋）-BmCPI／BmGAPDH和含非甲基化胞嘧啶和鸟嘌呤的寡聚脱氧核苷酸（CpGODN）免疫BALB／c小鼠，并设PBS对照组及空质粒对照组，共免疫3次，每次间隔2周。采用RT-PCR方法检测肌肉组织内的目的基因，采用四甲基偶氮唑盐（MTT）法和ELISA法分别检测免疫小鼠T淋巴细胞刺激增殖指数和血清中IFN-γ、IL-4水平。统计学分析采用t检验。结果pcDNA3．1（＋）-BmcPI／BmGAPDH复合基因真核表达载体免疫小鼠后，从小鼠肌肉组织内扩增出目的基因。免疫6周后，pcDNA3．1（＋）-BmCPI／CpG组和pcDNA3．1（＋）-BmCPI／BmGAPDH／CpG组T淋巴细胞刺激增殖指数分别为1．466±0．635和1．610±0．112，显著高于PBS组的1．004±0．019和pcDNA3．1（＋）-CpG组的1．078±0．129（t=64．438、45．318、42．749、34．314，均P〈0．05）。免疫4周后，pcDNA3．1（＋）-BmCPI／BmGAPDH／CpG组IFN-γ、IL-4水平均高于pcDNA3．1（＋）-CpG组（t=288．053、76．453，均P〈0．05），其中IFN-γ水平与pcDNA3．1（＋）-BmCPI／CpG组相比也有明显提高（t=129．642，P〈0．05）。结论pcDNA3．1（＋）-BmCPI／BmGAPDH重组复合基因真核表达载体能在小鼠体内表达，并可有效诱导特异性细胞免疫应答。Objective To construct the eukaryotic expression plasmids containing cysteine protease inhibitor （CPI） and glyceraldehydes-3-phosphate dehydrogenase （GAPDH） gene from periodic Brugia malayi （Bin）, and to observe its cellular immune response in mouse. Methods peDNA3. 1（＋）-BmCPI/BrnGAPDH was constructed. The recombinant plasmids were screened and identified by digestion with restriction enzyme. BALB/c mice were injected intramuscularly with a dosage of 100 μg purified recombinant plasmid DNA with GpG oligodeoxynucleotide （CpG ODN） and two same doses were administrated at 2-week intervals, pcDNA3. 1 （＋） and phosphate buffered solution （PBS） were used as controls. The tissue of muscles at 4 weeks after the third injection was collected and the target gene was detected by reverse transcription-polymerase chain reaction （RTPCR）. Two weeks after the third immunization, the stimulation index （SI） of spleen lymphocytes of immunized mice was measured by methylthiazolyldiphenyl-tetrazolium bromide （MTT） method and the serum levels of interleukin （IL）-4 and interferon （IFN）-γ were detected by enzyme-linked immunosorbent assay （ELISA）. The data were analyzed by t test. Results BmCPI/BmGAPDH gene in the injected muscle of the immunized mice was detected by RT-PCR. At 6 weeks after immunization, the SI of spleen T lymphocytes in pcDNA3.1（＋）-BmCPI/CpG group and pcDNA3. 1 （＋）-BrnCPI/BmGAPDH/CpG group were 1. 466＋ 0. 635 and 1. 610 ＋ 0. 112, respectively, which were both higher than PBS group and pcDNA3. 1（＋）-CpG group （1. 004＋0. 019 and 1. 078＋0. 129, respectively） （t=64. 438, 45. 318, 42. 749 and 34. 314, respectively; all P〈0.05）. At 4 weeks after immunization, the serum levels of IL-4 and IFN-γ of mice in pcDNA3. 1 （＋）-BmCPI/BmGAPDH/ CpG group were significantly higher than those in peDNA3. 1 （＋）-CpG group （t = 288. 053 and 76. 453, respectively; both P〈0.05）, while the serum level of IFN-γ was also higher than th

关 键 词：丝虫 马来 半胱氨酸蛋白酶抑制剂 甘油醛-3-磷酸脱氢酶类 基因表达 遗传载体 质粒 

分 类 号：R383.16[医药卫生—医学寄生虫学]