汉坦病毒GM04-38株N-联糖基化位点在细胞融合中的作用
发布时间：2019-02-27 09:58
【摘要】： 目的N-联寡糖链可以在很多方面影响蛋白的功能。已有报道显示某些病毒融合蛋白的寡糖链缺失可使病毒的细胞融合功能缺陷或丢失。已有报道显示汉坦病毒的G2糖蛋白可能是其融合蛋白。鉴于汉坦病毒GM04-38株包膜糖蛋白G1和G2也是通过N-联糖基化作用来修饰的,本研究拟通过实验对汉坦病毒N-联糖基化位点在细胞融合中的作用进行初步探讨。从而为阐明HV的细胞融合机制、研制有效的汉坦病毒新型疫苗和治疗制剂奠定基础。 方法根据GM04-38株M片段cDNA基因序列应用Primer 5软件设计引物,同源重组PCR法构建5个单个氨基酸突变的N-联糖基化位点的克隆载体。PCR扩增N-联糖基化位点突变体的编码区基因,双酶切后与经过同样双酶切的表达载体pCAGGS/MCS连接,转化大肠杆菌DH5a,氨苄青霉素筛选N-联糖基化位点单个氨基酸突变体的表达载体。经双酶切和测序证实。将真核表达质粒G1Bgl, GmG2及4个G1突变体,1个G2突变体提取后分别共转染Vero E6细胞,间接免疫荧光检测蛋白表达情况,免疫印迹实验确定糖蛋白表达情况,并经酸性MEM处理、Giemsa染色后观察细胞融合现象的发生 结果1.构建了5个N-联糖基化位点单个氨基酸突变的克隆载体N134A、N235A、N347A、N399A、N928A双酶切鉴定载体长度约为2.7kb;N134A、N235A、N347A、N399A突变体长度约为2.1kb;N928A突变体长度约为1.6kb,并经测序证实。 2.构建了5个N-联糖基化位点单个氨基酸突变的表达载体G1Bgl-N134A、GlBgl-N235A、GlBgl-N347A、G1Bgl-N399A、GmG2-N928A、双酶切鉴定,载体长度为4.7kb;N134A、N235A、N347A、N399A突变体目的片段长度约为2.1kb;N928A突变体目的片段长度约为1.6kb,并经测序证实。 3.间接免疫荧光实验显示野毒株GlBgl与GmG2共转染组、G1Bgl及各N-联糖基化位点突变体转染组均有亮绿色荧光信号产生,呈胞浆分布。 4.免疫印迹实验显示野毒株GlBgl与GmG2共转染组出现两条带,分别为68KDa和55KDa,N134A突变体转染组不显示G1条带,其余各突变体转染组均显示G1、G2两条带。 5.共转染N134A-:或N928A突变体组未出现细胞融合现象,而其他突变体及野毒株GlBgl与GmG2共转染后则出现融合现象。 结论G1上的134位点可能与蛋白正确折叠有密切关系,134位点突变极可能导致蛋白的错误折叠,进而无法转运出内质网。G2上的928位点对细胞融合有重要作用,提示汉坦病毒融合肽很有可能位于G2上
[Abstract]:Objective N-oligosaccharides can affect the function of proteins in many ways. It has been reported that the lack of oligosaccharide chain of some viral fusion proteins may lead to the defect or loss of fusion function of virus cells. It has been reported that the G2 glycoprotein of Hantavirus may be its fusion protein. In view of the fact that envelope glycoprotein G1 and G2 of Hantavirus GM04-38 strain were also modified by N-linked glycosylation, the role of Hantavirus N-linked glycosylation sites in cell fusion was investigated in this study. It lays a foundation for elucidating the cell fusion mechanism of HV and developing an effective new vaccine and therapeutic preparation for Hantavirus. Methods according to the sequence of M fragment cDNA gene of GM04-38 strain, primers were designed by Primer 5 software. Five single amino acid mutated N-linked glycosylation sites were constructed by homologous recombination PCR method. The coding region genes of N-linked glycosylation site mutants were amplified by polymerase chain reaction (PCR) and ligated with the expression vector pCAGGS/MCS with the same double enzyme digestion. The expression vector of single amino acid mutants of N-linked glycosylation site was screened by transforming E. coli DH5a, ampicillin. It was confirmed by double enzyme digestion and sequencing. The eukaryotic expression plasmid G1BGL, GmG2 and four G1 mutants and one G2 mutant were extracted and co-transfected into Vero E6 cells. The protein expression was detected by indirect immunofluorescence assay. The expression of glycoprotein was determined by immunoblotting and treated with acidic MEM. Observation of cell fusion after Giemsa staining 1. Five single amino acid mutants N134A, N235A, N347A, N399A, N928A, N235A, N347A, N399A were constructed. The length of N134A, N347A and N399A was about 2.1kb, 2.7kb and 2.1kb, respectively. The length of the mutant N928A was about 1.6 kb, which was confirmed by sequencing. 2. Five single amino acid mutants of N-linked glycosylation sites were constructed. The expression vector G1BglGN134A, GlBglGN235A, GlBglass N347A, G1BglN399A, GmG2xN928A were identified by double enzyme digestion, and the length of the vector was 4.7 kb.The length of the vector was 4.7kb.The length of the vector was 4.7kb. The fragment length of N134A, N235A, N347A, N399A mutants was about 2.1 kbb / N928A mutants, and was confirmed by sequencing. 3. Indirect immunofluorescence assay showed that bright green fluorescence signals were produced in the co-transfection group of wild strain GlBgl and GmG2, G1Bgl and each N-linked glycosylation site mutant group, showing cytoplasmic distribution. 4. Western blot analysis showed that there were two bands in the co-transfection group of wild strain GlBgl and GmG2, which were 68KDa and 55KDa, respectively. There were no G1 bands in the N134A mutant transfection group, and G1 and G2 bands in the other mutants' transfection groups. 5. There was no fusion phenomenon in N134A: or N928A mutant group, but there was no fusion phenomenon after co-transfection of other mutants and wild strain GlBgl with GmG2. Conclusion the 134 site on G1 may be closely related to the correct folding of the protein, and the 134 mutation may lead to the misfolding of the protein, thus unable to transport out of the endoplasmic reticulum. The 928 site on G2 may play an important role in cell fusion. It is suggested that the fusion peptide of Hantavirus is probably located on G2.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373
本文编号：2431350
[Abstract]:Objective N-oligosaccharides can affect the function of proteins in many ways. It has been reported that the lack of oligosaccharide chain of some viral fusion proteins may lead to the defect or loss of fusion function of virus cells. It has been reported that the G2 glycoprotein of Hantavirus may be its fusion protein. In view of the fact that envelope glycoprotein G1 and G2 of Hantavirus GM04-38 strain were also modified by N-linked glycosylation, the role of Hantavirus N-linked glycosylation sites in cell fusion was investigated in this study. It lays a foundation for elucidating the cell fusion mechanism of HV and developing an effective new vaccine and therapeutic preparation for Hantavirus. Methods according to the sequence of M fragment cDNA gene of GM04-38 strain, primers were designed by Primer 5 software. Five single amino acid mutated N-linked glycosylation sites were constructed by homologous recombination PCR method. The coding region genes of N-linked glycosylation site mutants were amplified by polymerase chain reaction (PCR) and ligated with the expression vector pCAGGS/MCS with the same double enzyme digestion. The expression vector of single amino acid mutants of N-linked glycosylation site was screened by transforming E. coli DH5a, ampicillin. It was confirmed by double enzyme digestion and sequencing. The eukaryotic expression plasmid G1BGL, GmG2 and four G1 mutants and one G2 mutant were extracted and co-transfected into Vero E6 cells. The protein expression was detected by indirect immunofluorescence assay. The expression of glycoprotein was determined by immunoblotting and treated with acidic MEM. Observation of cell fusion after Giemsa staining 1. Five single amino acid mutants N134A, N235A, N347A, N399A, N928A, N235A, N347A, N399A were constructed. The length of N134A, N347A and N399A was about 2.1kb, 2.7kb and 2.1kb, respectively. The length of the mutant N928A was about 1.6 kb, which was confirmed by sequencing. 2. Five single amino acid mutants of N-linked glycosylation sites were constructed. The expression vector G1BglGN134A, GlBglGN235A, GlBglass N347A, G1BglN399A, GmG2xN928A were identified by double enzyme digestion, and the length of the vector was 4.7 kb.The length of the vector was 4.7kb.The length of the vector was 4.7kb. The fragment length of N134A, N235A, N347A, N399A mutants was about 2.1 kbb / N928A mutants, and was confirmed by sequencing. 3. Indirect immunofluorescence assay showed that bright green fluorescence signals were produced in the co-transfection group of wild strain GlBgl and GmG2, G1Bgl and each N-linked glycosylation site mutant group, showing cytoplasmic distribution. 4. Western blot analysis showed that there were two bands in the co-transfection group of wild strain GlBgl and GmG2, which were 68KDa and 55KDa, respectively. There were no G1 bands in the N134A mutant transfection group, and G1 and G2 bands in the other mutants' transfection groups. 5. There was no fusion phenomenon in N134A: or N928A mutant group, but there was no fusion phenomenon after co-transfection of other mutants and wild strain GlBgl with GmG2. Conclusion the 134 site on G1 may be closely related to the correct folding of the protein, and the 134 mutation may lead to the misfolding of the protein, thus unable to transport out of the endoplasmic reticulum. The 928 site on G2 may play an important role in cell fusion. It is suggested that the fusion peptide of Hantavirus is probably located on G2.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373
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