弓形虫棒状体蛋白7真核表达载体的构建及其免疫保护作用

Construction of an eukaryotic vector of rhoptry protein 7 from Toxoplasma gondii and its immunoprotective effect

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 the eukaryotic expression vector of rhoptry protein 7（ ROP7） of Toxoplasma gondii and to evaluate the protective effect induced by recombinant plasmid on mice. Methods PCR primers were designed based on the ROP7 sequence of Toxoplasma gondii in Genbank. The ROP7 gene was amplified by PCR and inserted into the eukaryotic vector pEGFP-C1 and the recombinant plasmid pEGFP-C1 /ROP7（ pROP7） was constructed. Then the plasmids were transfected into HEK293 T cells to verify the expression in eukaryotic cells. A total of 36 female BALB /c mice were randomly divided into three groups： phosphate buffer saline（ PBS） control group,pEGFP-C1 control group and the pROP7 experimental group. Then PBS,pEGFP-C1 and pROP7 were used to immunize the corresponding group of mice respectively. The special serum IgG level was detected with ELISA and the survival time of challenged mice was recorded to evaluate the immune protection. Results The target gene（ 1 728 bp） was successfully amplified by PCR.The recombinant plasmid was successfully constructed and expressed in HEK293 T cells. Its protein was recognized with a goat anti-Toxopasma gondii antibody. Mice immunized with the recombinant plasmid produced high levels of serum antibodies.In the challenge experiment,the survival time of mice in pROP7 experience group was significantly prolonged compared with that of control groups（ P〈0. 05）. Conclusion With the immunological effect of this expressed recombinant plasmid,the present study might provide reference for future study of DNA vaccine against Toxoplasma gondii.