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目的构建弓形虫棒状体蛋白7(ROP7)基因的真核重组表达载体,观察其对小鼠的免疫保护作用。方法根据GenBank发表的弓形虫ROP7序列设计合成一对引物,通过PCR扩增ROP7基因,将其插入真核表达载体pEGFP-C1中构建重组质粒pEGFP-C1/ROP7(pROP7)。用重组质粒转染HEK293T细胞并验证其在细胞内的表达。将36只雌性BALB/c小鼠随机分为3组:磷酸缓冲盐溶液(PBS)对照组、pEGFP-C1对照组和pROP7实验组。然后用PBS、pEGFP-C1和pROP7分别免疫相应组别的小鼠。采用ELISA检测小鼠血清特异IgG水平,观察弓形虫感染小鼠后的存活时间并评价其免疫保护力。结果PCR扩增出1 728 bp的目的基因片段,真核表达载体构建成功,且能在HEK293T细胞中表达。目的基因的相应蛋白可被羊抗弓形虫多克隆抗体识别。重组质粒免疫的小鼠产生了较高水平的血清抗体。虫体攻击试验中,pROP7实验组小鼠的生存时间较对照组小鼠延长(P<0.05)。结论本研究构建的真核表达质粒在对抗弓形虫感染时具有一定的免疫保护作用,为弓形虫基因疫苗的研制提供了参考。
Objective To construct eukaryotic recombinant expression vector of Toxoplasma gondii Rod - like Protein 7 (ROP7) and observe its immunoprotective effect on mice. Methods A pair of primers was designed and synthesized according to the ROP7 sequence of Toxoplasma gondii published in GenBank. The ROP7 gene was amplified by PCR and inserted into the eukaryotic expression vector pEGFP-C1 to construct the recombinant plasmid pEGFP-C1 / ROP7 (pROP7). HEK293T cells were transfected with recombinant plasmids and their expression in cells was verified. 36 female BALB / c mice were randomly divided into 3 groups: phosphate buffered saline (PBS) control group, pEGFP-C1 control group and pROP7 experimental group. The corresponding group of mice were then immunized with PBS, pEGFP-C1 and pROP7, respectively. The level of serum specific IgG was detected by ELISA, the survival time of mice infected with Toxoplasma gondii was observed, and the immunoprotective power was evaluated. Results The target gene fragment of 1 728 bp was amplified by PCR. The eukaryotic expression vector was successfully constructed and expressed in HEK293T cells. The corresponding protein of the target gene can be identified by anti-T.gondii polyclonal antibody. Mice immunized with recombinant plasmids produced higher levels of serum antibodies. In the experiment of parasitological attack, the survival time of pROP7 mice was longer than that of the control mice (P <0.05). Conclusion The eukaryotic expression plasmid constructed in this study has certain immunoprotective effect against T. gondii infection and provides a reference for the development of Toxoplasma gondii gene vaccine.