汉坦病毒包膜糖蛋白基因表达载体的构建、表达与生物学活性分析
发布时间：2018-11-02 07:55
【摘要】： 目的构建汉坦病毒(Hantavirus,HV)GM04-38株包膜糖蛋基因G1、G2的真核表达载体,并将其在Vero E6细胞中进行表达,研究其表达特点,并且观察细胞融合现象的发生,为研究汉坦病毒基因结构与功能、病毒糖蛋白的结构与功能、研制有效的汉坦病毒基因工程亚单位疫苗奠定基础。构建汉坦病毒糖基化位点的突变体,对以后研究糖基化位点改变对细胞融合的影响具有重要意义。 方法根据GenBank SEO型HV M、S片段cDNA基因保守序列及相关参考文献设计引物,以质粒pUCm-M1和pMD18-M2为模板,应用PCR方法分别扩增HV糖蛋白G1、G2的全长基因,并在基因两端创建相应的酶切位点,将PCR产物和表达载体pCAGGS/MCS分别双酶切后连接,转化大肠杆菌DH5α,经氨苄筛选,酶切和测序鉴定正确后,两种载体分别命名为pCAGGS-G1、pCAGGS-G2,然后将两种糖蛋白载体共转染Vero E6细胞,酸性MEM处理、Giemsa染色后观察细胞融合现象的产生。同时用pCAGGS-NP与两种糖蛋白载体共表达,看核蛋白是否对糖蛋白的融合有增强作用,并以间接免疫荧光实验(indirect immunofluorescence assay,IIFA)来观察糖蛋白和核蛋白的表达情况。利用基因定点突变的方法构建了五个糖蛋白突变体,即将G1、G2上的的天冬酰胺置换为丙氨酸,根据被替换的位置,突变体分别命名为N134A、N235A、N347A、N399A、N928A。 结果1.构建了含有包膜糖蛋白基因G1、G2的真核表达载体pCAGGS-G1和pCAGGS-G2,双酶切鉴定目的片段分别为2.0kb和1.5kb,载体片段为4.7kb并测序证实。 2.间接免疫荧光试验显示糖蛋白组、核蛋白组及两者的共表达组皆有亮绿色荧光信号产生,呈胞浆分布。 3.糖蛋白G1、G2共转染后在偏酸性条件下可引起Vero E6细胞发生融合,而转染pCAGGS-NP的细胞则没有融合现象发生;将核蛋白与糖蛋白共表达后也未出现明显的促进效应。 4.构建了五个N-连糖基化位点的突变体,测序图谱显示原序列中的天冬酰胺(N)均被置换为丙氨酸(A)。 结论成功构建了汉坦病毒包膜糖蛋白基因G1、G2的真核表达载体并在Vero E6细胞中有效表达,二者的共表达可以产生良好的生物学活性,在酸性条件下引起Vero E6细胞发生融合。为进一步研究糖蛋白的结构与功能,制备有效的亚单位疫苗奠定了坚实基础。成功构建了各糖基化位点的突变体,为进一步研究N-连糖基化的缺失对细胞融合的影响提供了必要条件。
[Abstract]:Objective to construct the eukaryotic expression vector of G1G 2 of Hantavirus (Hantavirus,HV) GM04-38 strain, express it in Vero E6 cells, study its expression characteristics, and observe the occurrence of cell fusion. In order to study the structure and function of Hantavirus gene, the structure and function of virus glycoprotein, and to develop an effective vaccine of Hantavirus gene engineering subunit. The construction of the mutant of glycosylation site of Hantavirus is of great significance to study the effect of glycosylation site change on cell fusion in the future. Methods Primer was designed according to the conserved sequence of cDNA gene of GenBank SEO type HV map-S fragment and related references. Using plasmid pUCm-M1 and pMD18-M2 as templates, PCR method was used to amplify the full length of HV glycoprotein G1G 2 gene, respectively. The PCR product and the expression vector pCAGGS/MCS were digested and ligated respectively, then transformed into Escherichia coli DH5 伪. After screening with ampicillin, the two vectors were identified correctly by enzyme digestion and sequencing. The two vectors were named pCAGGS-G1, respectively. PCAGGS-G2, then co-transfected Vero E6 cells with two kinds of glycoprotein vectors. The cells were treated with acidic MEM, and the cell fusion was observed by Giemsa staining. At the same time, pCAGGS-NP was co-expressed with two kinds of glycoprotein vectors to see if the nucleoprotein enhanced the fusion of glycoprotein, and the expression of glycoprotein and nucleoprotein was observed by indirect immunofluorescence assay (indirect immunofluorescence assay,IIFA). Five glycoprotein mutants were constructed by site-directed mutagenesis. The asparagine on G1G 2 was replaced with alanine. According to the position of substitution, the mutants were named N134AN235AN347AN399AN399AN928A respectively. Result 1. The eukaryotic expression vector pCAGGS-G1 and pCAGGS-G2, were constructed and identified as 2.0kb and 1.5kb, respectively. The vector fragment was 4.7kb and confirmed by sequencing. 2. Indirect immunofluorescence assay showed that bright green fluorescent signals were produced in glycoprotein group, nucleoprotein group and co-expression group, and distributed in cytoplasm. 3. The fusion of Vero E6 cells was induced by co-transfection of glycoprotein G1G 1 / G 2, but no fusion was found in pCAGGS-NP transfected cells, nor did the coexpression of ribosin and glycoprotein. 4. Five mutants of N-glycosylation sites were constructed and sequenced to show that asparagine (N) in the original sequence was replaced with alanine (A). Conclusion the eukaryotic expression vector of Hantavirus envelope glycoprotein gene G1FG 2 was successfully constructed and effectively expressed in Vero E6 cells. The co-expression of the two genes can produce good biological activity and induce the fusion of Vero E6 cells under acidic conditions. It lays a solid foundation for the further study of the structure and function of glycoprotein and the preparation of effective subunit vaccine. The mutants of various glycosylation sites were successfully constructed, which provided the necessary conditions for further study of the effect of N-linked glycosylation on cell fusion.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
本文编号：2305406
[Abstract]:Objective to construct the eukaryotic expression vector of G1G 2 of Hantavirus (Hantavirus,HV) GM04-38 strain, express it in Vero E6 cells, study its expression characteristics, and observe the occurrence of cell fusion. In order to study the structure and function of Hantavirus gene, the structure and function of virus glycoprotein, and to develop an effective vaccine of Hantavirus gene engineering subunit. The construction of the mutant of glycosylation site of Hantavirus is of great significance to study the effect of glycosylation site change on cell fusion in the future. Methods Primer was designed according to the conserved sequence of cDNA gene of GenBank SEO type HV map-S fragment and related references. Using plasmid pUCm-M1 and pMD18-M2 as templates, PCR method was used to amplify the full length of HV glycoprotein G1G 2 gene, respectively. The PCR product and the expression vector pCAGGS/MCS were digested and ligated respectively, then transformed into Escherichia coli DH5 伪. After screening with ampicillin, the two vectors were identified correctly by enzyme digestion and sequencing. The two vectors were named pCAGGS-G1, respectively. PCAGGS-G2, then co-transfected Vero E6 cells with two kinds of glycoprotein vectors. The cells were treated with acidic MEM, and the cell fusion was observed by Giemsa staining. At the same time, pCAGGS-NP was co-expressed with two kinds of glycoprotein vectors to see if the nucleoprotein enhanced the fusion of glycoprotein, and the expression of glycoprotein and nucleoprotein was observed by indirect immunofluorescence assay (indirect immunofluorescence assay,IIFA). Five glycoprotein mutants were constructed by site-directed mutagenesis. The asparagine on G1G 2 was replaced with alanine. According to the position of substitution, the mutants were named N134AN235AN347AN399AN399AN928A respectively. Result 1. The eukaryotic expression vector pCAGGS-G1 and pCAGGS-G2, were constructed and identified as 2.0kb and 1.5kb, respectively. The vector fragment was 4.7kb and confirmed by sequencing. 2. Indirect immunofluorescence assay showed that bright green fluorescent signals were produced in glycoprotein group, nucleoprotein group and co-expression group, and distributed in cytoplasm. 3. The fusion of Vero E6 cells was induced by co-transfection of glycoprotein G1G 1 / G 2, but no fusion was found in pCAGGS-NP transfected cells, nor did the coexpression of ribosin and glycoprotein. 4. Five mutants of N-glycosylation sites were constructed and sequenced to show that asparagine (N) in the original sequence was replaced with alanine (A). Conclusion the eukaryotic expression vector of Hantavirus envelope glycoprotein gene G1FG 2 was successfully constructed and effectively expressed in Vero E6 cells. The co-expression of the two genes can produce good biological activity and induce the fusion of Vero E6 cells under acidic conditions. It lays a solid foundation for the further study of the structure and function of glycoprotein and the preparation of effective subunit vaccine. The mutants of various glycosylation sites were successfully constructed, which provided the necessary conditions for further study of the effect of N-linked glycosylation on cell fusion.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
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