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弓形虫侵入相关分子棒状体蛋白1基因重组质粒的克隆及其DNA免疫研究

Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity

摘要:

目的构建含编码弓形虫棒状体蛋白1基因的重组质粒,将该重组质粒DNA免疫小鼠,观察其所诱导的免疫保护性反应。方法用PCR技术从弓形图ZS2分离株的基因组DNA中扩增编码棒状体蛋白1(ROP1)的基因片段,重组入pUC18-ROP1中的ROP1外源基因片段经酶切,连接等反应,亚克隆入pcDNA3真核表达载体,再经含氨苄LB培养基筛选,酶切,PCR鉴定:获取阳性克隆并大量制备,肌注法DNA免疫BALB/C小鼠,每只鼠100?μg,两周后同量加强免疫一次,以pcDNA3空质粒及生理盐水组为对照。分别于加强免疫后30d,50d,70d,用MTT法测定小鼠睥脏NK细胞活性,T淋巴细胞增殖活性,间接免疫荧光法记数T细胞亚群及ELISA检测血清IgG抗体。结果从ZS2株基因DNA中扩增出特异的ROP1基因片段并经TaqI酶切鉴定:克隆成功pcROP1重组质粒。用该质粒免疫小鼠30天后,脾脏明显增大;三次测定结果免疫组脾T淋巴细胞增值活性明显高于生理盐水及空质粒对照组。NK细胞杀伤活性免疫组钧高于对照组。T细胞亚群,CD4+细胞数与对照组相比,无明显变化,而CD8+细胞数显著增高(P<0.05)。血清抗体IgG 70d内检测结果8,与对照相比,无明显增高;而免疫后90d检测,明显增高。抗体滴度为本:1∶100。结论构建成功弓形虫pcROP1重组质粒,免疫小鼠后,该质粒诱导小鼠产生细胞及体液免疫应答。

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abstracts:

Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.

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