弓形虫GJS株微线体蛋白3基因真核表达质粒的构建及表达
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摘要
弓形虫(Toxoplasma gondii)是一种专性细胞内寄生原虫,可感染包括人在内的所有哺乳动物。弓形虫病可导致严重的公共卫生问题和广泛的经济损失。弓形虫微线体蛋白3(Microneme protein 3, MIC3)是一种分泌型黏附素,黏附在宿主细胞和寄生虫的表面,在弓形虫的速殖子、缓殖子和子孢子期均能表达。具有作为诊断和免疫的双重潜在价值。
根据已发表的弓形虫RH株MIC3的基因序列设计引物,采用PCR技术从弓形虫GJS株基因组DNA中扩增MIC3基因,克隆到pMD18-T载体,经PCR、酶切及测序鉴定后,阳性重组质粒酶切并亚克隆到真核表达载体pcDNA3.1(+)后进行PCR、酶切及测序鉴定。将重组质粒转染BHK-21细胞。间接免疫荧光染色证明质粒DNA成功转染到细胞中并得以表达,通过Western-blot分析,细胞裂解液及上清样品中各有一条约39.2 ku的条带,可被山羊抗弓形虫高免血清所识别,大小与预测值相符。表明真核表达质粒pcDNA3-MIC3中的MIC3基因在BHK-21细胞中获得表达且表达产物具有抗原性。重组质粒pcDNA3-MIC3肌肉注射免疫BALB/c小鼠后,通过ELISA检测血清特异抗体;经腹腔攻击感染弓形虫GJS株速殖子,观察小鼠的生存时间。免疫组小鼠血清特异性抗体较对照组差异极显著(P<0.01);攻击感染后免疫组小鼠平均存活时间较对照组明显延长。表明该核酸疫苗具有较好的免疫原性,能诱导小鼠产生良好的免疫保护作用。本研究为弓形虫病诊断和疫苗研制提供了候选抗原。
Toxoplasma gondii is an obligate intracellular protozoan parasite that infects all warm- blooded animals including humans. Toxoplasmosis causes serious public health problems and is of great economic importance worldwide. The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite which is expressed in tachyzoites, bradyzoites and sporozoites. MIC3 has two potential valuble applycation for general research, including diagnosis and immunity.
One pair of primers was designed based on the MIC3 gene sequences of RH strain Toxoplasma gondii in GenBank. The gene fragment encoding MIC3 was amplified by PCR from the genomic DNA of Toxoplasma gondii GJS strain and cloned into the vector pMD18-T. After PCR, restrictive digestion and sequencing identification, the positive plasmid was digested and subcloned into an eukaryotic expression vector pcDNA3.1 (+). The eukaryotic expression recombinant plasmid pcDNA3-MIC3 was identified by PCR, restriction enzyme digestion and sequencing analysis. Then, the pcDNA3-MIC3 plamid was transfected into BHK-21 cells mediated by Lipofectamine 2000. Indirect immunofluorescence showed that the recombinant plamid was expressed in BHK-21 cells. Western-blot analysis showed that a band approximately 39.2 ku in size in BHK-21 cells lysate and supernatant of the culture medium were recognized by the goat serum against Toxoplasma gondii, indicating that the MIC3 protein expressed transiently in the cells had antigenicity. After mice were injected with the recombinant plasmid pcDNA3-MIC3, the sera antibody were detected by indirect ELISA and survival time of mice after challenge with tachyzoites of Toxoplasma gondii GJS strain were observed. The results showed that the recombinant plasmid pcDNA3-MIC3 was successfully constructed and the immunized mice showed higher specific antibody level (P<0.01) than control. The survival time of vaccined mice was longer than that of control groups. It indicated that the nucleic acid vaccine had good immunity and could induce the immunoprotection in mice.
根据已发表的弓形虫RH株MIC3的基因序列设计引物,采用PCR技术从弓形虫GJS株基因组DNA中扩增MIC3基因,克隆到pMD18-T载体,经PCR、酶切及测序鉴定后,阳性重组质粒酶切并亚克隆到真核表达载体pcDNA3.1(+)后进行PCR、酶切及测序鉴定。将重组质粒转染BHK-21细胞。间接免疫荧光染色证明质粒DNA成功转染到细胞中并得以表达,通过Western-blot分析,细胞裂解液及上清样品中各有一条约39.2 ku的条带,可被山羊抗弓形虫高免血清所识别,大小与预测值相符。表明真核表达质粒pcDNA3-MIC3中的MIC3基因在BHK-21细胞中获得表达且表达产物具有抗原性。重组质粒pcDNA3-MIC3肌肉注射免疫BALB/c小鼠后,通过ELISA检测血清特异抗体;经腹腔攻击感染弓形虫GJS株速殖子,观察小鼠的生存时间。免疫组小鼠血清特异性抗体较对照组差异极显著(P<0.01);攻击感染后免疫组小鼠平均存活时间较对照组明显延长。表明该核酸疫苗具有较好的免疫原性,能诱导小鼠产生良好的免疫保护作用。本研究为弓形虫病诊断和疫苗研制提供了候选抗原。
Toxoplasma gondii is an obligate intracellular protozoan parasite that infects all warm- blooded animals including humans. Toxoplasmosis causes serious public health problems and is of great economic importance worldwide. The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite which is expressed in tachyzoites, bradyzoites and sporozoites. MIC3 has two potential valuble applycation for general research, including diagnosis and immunity.
One pair of primers was designed based on the MIC3 gene sequences of RH strain Toxoplasma gondii in GenBank. The gene fragment encoding MIC3 was amplified by PCR from the genomic DNA of Toxoplasma gondii GJS strain and cloned into the vector pMD18-T. After PCR, restrictive digestion and sequencing identification, the positive plasmid was digested and subcloned into an eukaryotic expression vector pcDNA3.1 (+). The eukaryotic expression recombinant plasmid pcDNA3-MIC3 was identified by PCR, restriction enzyme digestion and sequencing analysis. Then, the pcDNA3-MIC3 plamid was transfected into BHK-21 cells mediated by Lipofectamine 2000. Indirect immunofluorescence showed that the recombinant plamid was expressed in BHK-21 cells. Western-blot analysis showed that a band approximately 39.2 ku in size in BHK-21 cells lysate and supernatant of the culture medium were recognized by the goat serum against Toxoplasma gondii, indicating that the MIC3 protein expressed transiently in the cells had antigenicity. After mice were injected with the recombinant plasmid pcDNA3-MIC3, the sera antibody were detected by indirect ELISA and survival time of mice after challenge with tachyzoites of Toxoplasma gondii GJS strain were observed. The results showed that the recombinant plasmid pcDNA3-MIC3 was successfully constructed and the immunized mice showed higher specific antibody level (P<0.01) than control. The survival time of vaccined mice was longer than that of control groups. It indicated that the nucleic acid vaccine had good immunity and could induce the immunoprotection in mice.
引文
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