漢坦病毒GM04-38株N-聯(lián)糖基化位點(diǎn)在細(xì)胞融合中的作用
發(fā)布時(shí)間：2019-02-27 09:58
【摘要】： 目的N-聯(lián)寡糖鏈可以在很多方面影響蛋白的功能。已有報(bào)道顯示某些病毒融合蛋白的寡糖鏈缺失可使病毒的細(xì)胞融合功能缺陷或丟失。已有報(bào)道顯示漢坦病毒的G2糖蛋白可能是其融合蛋白。鑒于漢坦病毒GM04-38株包膜糖蛋白G1和G2也是通過N-聯(lián)糖基化作用來(lái)修飾的,本研究擬通過實(shí)驗(yàn)對(duì)漢坦病毒N-聯(lián)糖基化位點(diǎn)在細(xì)胞融合中的作用進(jìn)行初步探討。從而為闡明HV的細(xì)胞融合機(jī)制、研制有效的漢坦病毒新型疫苗和治療制劑奠定基礎(chǔ)。 方法根據(jù)GM04-38株M片段cDNA基因序列應(yīng)用Primer 5軟件設(shè)計(jì)引物,同源重組PCR法構(gòu)建5個(gè)單個(gè)氨基酸突變的N-聯(lián)糖基化位點(diǎn)的克隆載體。PCR擴(kuò)增N-聯(lián)糖基化位點(diǎn)突變體的編碼區(qū)基因,雙酶切后與經(jīng)過同樣雙酶切的表達(dá)載體pCAGGS/MCS連接,轉(zhuǎn)化大腸桿菌DH5a,氨芐青霉素篩選N-聯(lián)糖基化位點(diǎn)單個(gè)氨基酸突變體的表達(dá)載體。經(jīng)雙酶切和測(cè)序證實(shí)。將真核表達(dá)質(zhì)粒G1Bgl, GmG2及4個(gè)G1突變體,1個(gè)G2突變體提取后分別共轉(zhuǎn)染Vero E6細(xì)胞,間接免疫熒光檢測(cè)蛋白表達(dá)情況,免疫印跡實(shí)驗(yàn)確定糖蛋白表達(dá)情況,并經(jīng)酸性MEM處理、Giemsa染色后觀察細(xì)胞融合現(xiàn)象的發(fā)生 結(jié)果1.構(gòu)建了5個(gè)N-聯(lián)糖基化位點(diǎn)單個(gè)氨基酸突變的克隆載體N134A、N235A、N347A、N399A、N928A雙酶切鑒定載體長(zhǎng)度約為2.7kb;N134A、N235A、N347A、N399A突變體長(zhǎng)度約為2.1kb;N928A突變體長(zhǎng)度約為1.6kb,并經(jīng)測(cè)序證實(shí)。 2.構(gòu)建了5個(gè)N-聯(lián)糖基化位點(diǎn)單個(gè)氨基酸突變的表達(dá)載體G1Bgl-N134A、GlBgl-N235A、GlBgl-N347A、G1Bgl-N399A、GmG2-N928A、雙酶切鑒定,載體長(zhǎng)度為4.7kb;N134A、N235A、N347A、N399A突變體目的片段長(zhǎng)度約為2.1kb;N928A突變體目的片段長(zhǎng)度約為1.6kb,并經(jīng)測(cè)序證實(shí)。 3.間接免疫熒光實(shí)驗(yàn)顯示野毒株GlBgl與GmG2共轉(zhuǎn)染組、G1Bgl及各N-聯(lián)糖基化位點(diǎn)突變體轉(zhuǎn)染組均有亮綠色熒光信號(hào)產(chǎn)生,呈胞漿分布。 4.免疫印跡實(shí)驗(yàn)顯示野毒株GlBgl與GmG2共轉(zhuǎn)染組出現(xiàn)兩條帶,分別為68KDa和55KDa,N134A突變體轉(zhuǎn)染組不顯示G1條帶,其余各突變體轉(zhuǎn)染組均顯示G1、G2兩條帶。 5.共轉(zhuǎn)染N134A-:或N928A突變體組未出現(xiàn)細(xì)胞融合現(xiàn)象,而其他突變體及野毒株GlBgl與GmG2共轉(zhuǎn)染后則出現(xiàn)融合現(xiàn)象。 結(jié)論G1上的134位點(diǎn)可能與蛋白正確折疊有密切關(guān)系,134位點(diǎn)突變極可能導(dǎo)致蛋白的錯(cuò)誤折疊,進(jìn)而無(wú)法轉(zhuǎn)運(yùn)出內(nèi)質(zhì)網(wǎng)。G2上的928位點(diǎn)對(duì)細(xì)胞融合有重要作用,提示漢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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373
本文編號(hào)：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373
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