弓形虫pVAX1-SAG2真核表达质粒的构建及其诱导的小鼠免疫应答

目的　构建弓形虫主要速殖子表面抗原 2 (SAG2 )编码基因真核表达质粒pVAX1-SAG2 ,并接种小鼠 ,分析其所诱导的免疫应答。方法　以限制性内切酶EcoRⅠ与KpnⅠ双酶切从重组质粒 pGEM/SAG2中获得SAG2目的基因片段 ,约5 92个bp ,将其插入真核表达载体 pVAX1多克隆位点 ,构建重组质粒 pVAX1-SAG2 ,并转化大肠杆菌JM 10 9,阳性克隆以双酶切与PCR法鉴定。大量提取纯化重组质粒 pVAX1-SAG2 ,5 0 μg肌肉注射小鼠左后腿内侧肌肉 ,3周后加强免疫一次 ;RT-PCR检测SAG2在小鼠肌肉中的转录表达 ,流式细胞仪测定T细胞亚群 ,以速殖子虫体抗原作ELISA测定小鼠血清IgG抗体。结果　真核表达重组质粒 pVAX1-SAG2双酶切鉴定及PCR扩增结果与预期结果相符合。RT -PCR从注射部位肌肉组织总RNA中扩增出SAG2目的基因条带 ;重组质粒 pVAX1-SAG2免疫组CD+ 4 细胞数为 32 .35± 5 .38,显著高于空质粒pVAX1及生理盐水 (NS)二对照组 (P <0 .0 1) ,后二者CD+ 4 细胞数分别为 19.6 5± 4 .2 1与 17.84± 1.5 9;免疫组CD+ 8细胞数为 18.6 7± 2 .37,但与空质粒及NS对照组相比 ,差别不显著 (P >0 .0 5 )。ELISA测定结果显示 pVAX1/SAG2免疫组小鼠血清中出现抗弓形虫特异性IgG抗体。结论　构建成功SAG2真核表达重组质粒 pVAX1-SAG2 ,其能在

Construction of pVAX1-SAG2 Plasmid Encoding the SAG2 Protein of Toxoplasma Gondii and the Immnune Responses Induced in Mice

To construct the pVAX1-SAG2 recombinant plasmid encoding the SAG2 protein of Toxoplasma gondii and evaluate the immune responses in mice as a DNA vaccine.The SAG2 encoding DNA fragment was obtained from the plasmid pGEM/SAG2 by double enzymes digestion,and inserted into the poly-linker of eukaryotic expression plasmid pVAX1 to construct recombinant pVAX1-SAG2,which was transferred into the competent cell E.coli JM109;Positive bacterial clones contain the recombinants was identified and the recombinant plasmid pVAX1-SAG2 was extracted and purified from E.coli 10 9 in large scale for DNA vaccine.The experimental mice was inoculated by intramuscular injection with 50μg pVAX1-SAG2 plasmid,pVAX1 plasmid and normal saline was used as controls.A booster dose was given 3 weeks later.The expression of SAG2 in muscles was detected by RT-PCR,and the numbers of CD +_4/CD +_8 T cells andthe specific IgG level in sera of mice were determined by direct immunofluorescent assay and ELISA assay respectively.The recombinant plasmid pVAX1-SAG2 was constructed and identified containing the target SAG2 DNA fragment.And the SAG2 specific DNA fragment was amplified from the RNA extracts of the muscular tissue injected with plasmid pVAX1-SAG2 by RT-PCR.It was found that the number of CD +_4 T cells was significant increased in the mice group inoculated with pVAX1-SAG2 as compared with those on the control groups inoculated with pVAX1 and NS(P<0.01),but the change on the number of CD +_8 T cells was insignificant among the experiment groups(P>0.05).Sera of the pVAX1/SAG2 immunized mice exhibited IgG antibody to Toxoplasma gondii tachyzoites antigen.It is concluded that the recombinant plasmid pVAX1/SAG2 encoding the SAG2 protein of Toxoplasma gondii was constructed,that can both induced humoral and cellular immune responses in mice as a DNA vaccine.