弓形虫GJS株微线体蛋白3基因的克隆、表达及鉴定
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摘要
弓形虫(Toxoplasma gondii)为专性细胞内寄生原虫,呈全球分布,引起的弓形虫病是一种严重的人兽共患的寄生虫病,对人类健康和畜牧业生产造成了严重危害。弓形虫病诊断方法和免疫疫苗已成为弓形虫研究的当务之急。微线体蛋白3(Microneme protein3 MIC3)为弓形虫生活史各期均分泌的抗原,具有高度的保守性和免疫原性,作为疫苗候选因子受到人们的关注。
根据已发表的弓形虫RH株MIC3的基因序列设计引物,利用PCR技术从弓形虫GJS株基因组中扩增到MIC3基因,通过将产物与pMD-18T载体连接,构建重组质粒pMD-MIC3。基因测序结果表明,本试验所获得的MIC3基因核苷酸序列和所编码氨基酸的序列与GenBank中收录的标准强毒RH株的同源性分别为99.7%和99.2 %。根据MIC3开放阅读框(ORF)序列重新设计一对去掉信号肽编码序列的引物,以重组质粒pMD-MIC3为模板,进行PCR扩增,将扩增产物与pET-30a表达质粒连接,构建了重组原核表达载体pET- MIC3,将其转化到BL21(DE3)宿主菌中,用IPTG进行诱导表达,所表达的目的蛋白为包涵体。SDS-PAGE和Western-blot分析表明,MIC3基因在大肠杆菌中以包涵体的形式成功获得了表达,分子量大小为44 ku左右,与预期的大小一致,而且可被猪抗弓形虫阳性血清所识别。通过裂解、洗涤、变性、复性等方法对包涵体蛋白进行处理,以电泳切胶回收所获得的纯化产物作为抗原,免疫小鼠制备抗重组蛋白血清。用弓形虫代谢分泌抗原(ESA)和重组抗原分别对抗血清进行间接ELISA检测,小鼠免疫后产生了较高水平的特异性抗体,表明MIC3重组蛋白具有较好的免疫原性。本研究为弓形虫病免疫诊断和疫苗研制提供了候选抗原。
Toxoplasma gondii is an obligate intracellular parasite that infects a wide range of hosts, including human and domesticated animals throughout the world.So the toxoplasmsis can harm to people’s health and cause considerable economic loss in the farming industry. The development of an effective vaccine against Toxoplasma gondii would be of great value to both human and veterinary medicine., due to microneme 3(MIC3) antigen is secreted by the microneme of tachyzoites and bradyzoites. They are highly conservative and immunogenic, were selected as antigens for diagnosis and vaccination totoxoplasmosis.
In the present study, a pair of primers was designed based on the MIC3 gene sequences of RH strain Toxoplasma gondii in GenBank. The MIC3 gene was amplified by PCR from genomic DNA of GJS strain Toxoplasma gondii, and cloned into pMD-18T vector to construct the recombinant plasmid pMD-MIC3. The result showed that the identities of the cloned MIC3 gene with the data published in GenBank in nucleotide and amino acid sequence were 99.7% and 99.2%, respectively. After then, Primers without signal peptide were designed according to the ORF sequence of the MIC3 gene, and the pMD-MIC3 recombinant plasmid used as template in the PCR reaction, the amplified products were ligated to the pET-30a vector, and transformed into BL21(DE3) host cells. Expression of the recombinant plasmid was induced with IPTG, and resulted in a high level of protein expression. The expressed products were inclusion body. SDS-PAGE experiments revealed that fusion protein’s molecular weight is approximately 44kD. Western-blot analysis showed that the expressed recombinant protein could be recognized by pig anti-Tg. positive serum.
The recombination fusion protein was purified by lysis, washing, denaturation and renaturation. The specific band of expression was excised from the gel and used to immunize mice .The antisera were collected from the immunized mice ,examined with Excreted-secreted antigen(ESA) and recomident antigent by ELISA respectively .The result show that the sensitivity and specificity of recomident antigent were more ideal. This also provid theory and experiment data to search for new recombiant antigent and produce multi-molecule vaccine.
根据已发表的弓形虫RH株MIC3的基因序列设计引物,利用PCR技术从弓形虫GJS株基因组中扩增到MIC3基因,通过将产物与pMD-18T载体连接,构建重组质粒pMD-MIC3。基因测序结果表明,本试验所获得的MIC3基因核苷酸序列和所编码氨基酸的序列与GenBank中收录的标准强毒RH株的同源性分别为99.7%和99.2 %。根据MIC3开放阅读框(ORF)序列重新设计一对去掉信号肽编码序列的引物,以重组质粒pMD-MIC3为模板,进行PCR扩增,将扩增产物与pET-30a表达质粒连接,构建了重组原核表达载体pET- MIC3,将其转化到BL21(DE3)宿主菌中,用IPTG进行诱导表达,所表达的目的蛋白为包涵体。SDS-PAGE和Western-blot分析表明,MIC3基因在大肠杆菌中以包涵体的形式成功获得了表达,分子量大小为44 ku左右,与预期的大小一致,而且可被猪抗弓形虫阳性血清所识别。通过裂解、洗涤、变性、复性等方法对包涵体蛋白进行处理,以电泳切胶回收所获得的纯化产物作为抗原,免疫小鼠制备抗重组蛋白血清。用弓形虫代谢分泌抗原(ESA)和重组抗原分别对抗血清进行间接ELISA检测,小鼠免疫后产生了较高水平的特异性抗体,表明MIC3重组蛋白具有较好的免疫原性。本研究为弓形虫病免疫诊断和疫苗研制提供了候选抗原。
Toxoplasma gondii is an obligate intracellular parasite that infects a wide range of hosts, including human and domesticated animals throughout the world.So the toxoplasmsis can harm to people’s health and cause considerable economic loss in the farming industry. The development of an effective vaccine against Toxoplasma gondii would be of great value to both human and veterinary medicine., due to microneme 3(MIC3) antigen is secreted by the microneme of tachyzoites and bradyzoites. They are highly conservative and immunogenic, were selected as antigens for diagnosis and vaccination totoxoplasmosis.
In the present study, a pair of primers was designed based on the MIC3 gene sequences of RH strain Toxoplasma gondii in GenBank. The MIC3 gene was amplified by PCR from genomic DNA of GJS strain Toxoplasma gondii, and cloned into pMD-18T vector to construct the recombinant plasmid pMD-MIC3. The result showed that the identities of the cloned MIC3 gene with the data published in GenBank in nucleotide and amino acid sequence were 99.7% and 99.2%, respectively. After then, Primers without signal peptide were designed according to the ORF sequence of the MIC3 gene, and the pMD-MIC3 recombinant plasmid used as template in the PCR reaction, the amplified products were ligated to the pET-30a vector, and transformed into BL21(DE3) host cells. Expression of the recombinant plasmid was induced with IPTG, and resulted in a high level of protein expression. The expressed products were inclusion body. SDS-PAGE experiments revealed that fusion protein’s molecular weight is approximately 44kD. Western-blot analysis showed that the expressed recombinant protein could be recognized by pig anti-Tg. positive serum.
The recombination fusion protein was purified by lysis, washing, denaturation and renaturation. The specific band of expression was excised from the gel and used to immunize mice .The antisera were collected from the immunized mice ,examined with Excreted-secreted antigen(ESA) and recomident antigent by ELISA respectively .The result show that the sensitivity and specificity of recomident antigent were more ideal. This also provid theory and experiment data to search for new recombiant antigent and produce multi-molecule vaccine.
引文
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