TgMAPK1核酸疫苗对小鼠的免疫保护性研究

Immunoprotective effect of a TgMAPK1 nucleic acid vaccine in mice

目的构建重组质粒pEGFP-MAPK1,制备弓形虫MAPK1核酸疫苗(DNA疫苗),评价该疫苗在小鼠体内引起的免疫效应以及在抗弓形虫感染过程中的保护作用。方法以弓形虫RH株cDNA作为PCR的模板扩增目的基因TgMAPK1,与真核表达载体pEGFP-C1连接,构建重组质粒pEGFP-MAPK1。pEGFP-MAPK1转染HEK293T细胞,通过荧光显微镜观察及Western blot检测其在真核细胞内的表达情况。将BALB/c雌性小鼠随机分成3组(PBS对照组、pEGFP-C1对照组和pEGFP-MAPK1实验组),大提质粒并免疫接种每组小鼠,ELISA检测每组小鼠在免疫接种前后血清抗体和脾细胞培养上清中细胞因子的变化。将弓形虫速殖子经腹腔注射入小鼠体内并每天记录小鼠的存活时间,评价该疫苗诱导产生的免疫保护效果。结果 PCR扩增出约1 599bp的目的基因片段,重组真核质粒pEGFPMAPK1构建成功。重组质粒和空质粒转染的细胞在荧光显微镜下均可观察到绿色荧光,提取的细胞总蛋白经Western blot检出能与相应抗体反应的分子质量单位约为58×103的目的蛋白。pEGFP-MAPK1免疫组小鼠较对照组能诱导产生更高水平的IgG(A值0.75~0.79)、IgG2a(A值0.65~0.71)和IFN-γ(725.67±23.25)pg/ml,差异有统计学意义(P<0.05),而IgG1、IL-4和IL-10各组间差异无统计学意义(P>0.05)。与对照组(6d)相比,pEGFP-MAPK1组小鼠(18d)感染弓形虫后的存活时间显著延长(P<0.05)。结论 TgMAPK1核酸疫苗能诱导小鼠产生较强的体液免疫反应和Th1型细胞免疫反应,表明该疫苗具有一定的抗弓形虫感染免疫保护作用,TgMAPK1蛋白可作为弓形虫疫苗候选抗原。

Objectives To construct the recombinant plasmid pEGFP-MAPK1 and to prepare a MAPK1 nucleic acid vaccine（DNA vaccine）against T.gondii in order to evaluate the immunoprotective effect of the vaccine against T.gondii in BALB/c mice. Methods cDNA of the T.gondii RH strain was used as a template in PCR.The TgMAPK1 gene was amplified using PCR and ligated into the eukaryotic expression vector pEGFP-C1.The recombinant plasmid pEGFPMAPK1 was constructed.pEGFP-MAPK1 was transfected into HEK293 Tcells.Plasmid expression in eukaryotes was verified with fluorescence microscopy and Western blotting.BALB/c female mice were randomly divided into three groups（a PBS control group,apEGFP-C1 control group,and a pEGFP-MAPK1 experimental group）.Plasmids were extracted using an endotoxin-free kit,and mice in each group were immunized with extracted plasmids.ELISA was used to detect changes in antibodies in sera and cytokines in cultured splenocytes from mice.T.gondii tachyzoites were intraperitoneally injected into mice,and the survival time of challenged mice was recorded daily to evaluate the immunoprotective effect of the MAPK1 DNA vaccine. Results The target gene（1,599 bp）was amplified using PCR,and the recombinant plasmid pEGFP-MAPK1 was successfully constructed.Cells transfected with the recombinant plasmid and an empty plasmid were observed under a fluorescence microscope.The recombinant protein had a molecular weight of about 58×10-3 and was recognized by goat polyclonal antibodies against T.gondii according to Western blotting.Mice immunized with pEGFP-MAPK1 had higher levels of IgG（A=0.75-0.79）,IgG2 a（A=0.65-0.71）,and IFN-γ（725.67±23.25 pg/ml）than levels in the control groups（P〈0.05）,while IgG1,IL-4,and IL-10 levels did not differ significantly between the groups（P〉0.05）.mice infected with T.gondii in the pEGFP-MAPK1 experimental group（18 d）had a significantly longer survival time（P〈0.05）than that of mice in the two control groups（6 d）. Conclusion The TgMAPK1 nucleic acid vaccine induced a strong humoral immune response and Th1 cell immune response in BALB/c mice,indicating that the vaccine triggered an immunoprotective effect against a T.gondii challenge.The TgMAPK1 protein can serve as a potential antigen with which to develop vaccines against T.gondii.