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弓形虫SAG1-GRA1复合基因真核表达重组质粒的构建与表达 被引量:2
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作者 綦廷娜 石畅 +2 位作者 汪政勇 宗永丽 李金福 《贵州医科大学学报》 CAS 2020年第3期286-291,共6页
目的:构建弓形虫SAG1-GRA1复合基因真核表达重组质粒,并观察其在小鼠胚胎成纤维细胞系NIH3T3细胞中的表达。方法:采用聚合酶链反应(PCR)法分别扩增弓形虫SAG1和GRA1基因片段,导入质粒载体pc DNA3.1(+),构建真核表达重组质粒pc DNA3.1(+)... 目的:构建弓形虫SAG1-GRA1复合基因真核表达重组质粒,并观察其在小鼠胚胎成纤维细胞系NIH3T3细胞中的表达。方法:采用聚合酶链反应(PCR)法分别扩增弓形虫SAG1和GRA1基因片段,导入质粒载体pc DNA3.1(+),构建真核表达重组质粒pc DNA3.1(+)-SAG1和pc DNA3.1(+)-GRA1,继而构建重组质粒pc DNA3.1(+)-SAG1-GRA1;设pc DNA3.1(+)-SAG1-GRA1为实验组、pc DNA3.1(+)为对照组分别转染NIH3T3细胞,采用免疫荧光染色法鉴定其在NIH3T3细胞中的表达。结果:限制性内切酶双酶切及DNA测序分析结果显示,插入pc DNA3.1(+)的片段分别为1008 bp、570 bp和1578 bp,与预期的SAG1、GRA1和SAG1-GRA1复合基因分子大小相当、序列一致;免疫荧光染色法结果显示,转染重组质粒pc DNA3.1(+)-SAG1-GRA1的NIH3T3细胞内观察到较强的绿色荧光,而转染pc DNA3.1(+)的NIH3T3细胞内未见绿色荧光。结论:成功构建了弓形虫SAG1-GRA1复合基因真核表达重组质粒pc DNA3.1(+)-SAG1-GRA1,且该重组质粒携带的SAG1-GRA1复合基因在NIH3T3细胞中获得表达。 展开更多
关键词 弓形虫属 sag1基因 GRA1基因 基因重组 重组质粒 体外表达
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Protective effect of DNA-mediated immunization with a combination of SAG1 and IL-2 gene adjuvant against infection of Toxoplasma gondii in mice
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作者 陈观今 陈海峰 +1 位作者 郭虹 郑焕钦 《Chinese Medical Journal》 SCIE CAS CSCD 2002年第10期8-12,142,共6页
To characterize the immune response induced by SAG1 encoding plasmid combined with IL 2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis Methods Mice were co inje... To characterize the immune response induced by SAG1 encoding plasmid combined with IL 2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis Methods Mice were co injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL 2 expression vector at a dose of 100 μg Booster immunizations were employed 2 more times at 3 week interval As controls, mice were inoculated with PBS or empty plasmid pcDNA3 Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN γ, as well as IL 4 To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally Results Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid With respect to the IgG isotype, co inoculation of IL 2 expression plasmid enhanced the level of IgG2a and the production of IFN γ Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival Conclusion Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co inoculation with IL 2 expression plasmid The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T gondii infection warrants further investigation 展开更多
关键词 DNA immunization · Toxoplasma gondi · surface antigen 1 (sag1) · gene adjuvant · IL 2
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