漢坦病毒包膜糖蛋白基因表達載體的構(gòu)建、表達與生物學(xué)活性分析
發(fā)布時間：2018-11-02 07:55
【摘要】： 目的構(gòu)建漢坦病毒(Hantavirus,HV)GM04-38株包膜糖蛋基因G1、G2的真核表達載體,并將其在Vero E6細胞中進行表達,研究其表達特點,并且觀察細胞融合現(xiàn)象的發(fā)生,為研究漢坦病毒基因結(jié)構(gòu)與功能、病毒糖蛋白的結(jié)構(gòu)與功能、研制有效的漢坦病毒基因工程亞單位疫苗奠定基礎(chǔ)。構(gòu)建漢坦病毒糖基化位點的突變體,對以后研究糖基化位點改變對細胞融合的影響具有重要意義。 方法根據(jù)GenBank SEO型HV M、S片段cDNA基因保守序列及相關(guān)參考文獻設(shè)計引物,以質(zhì)粒pUCm-M1和pMD18-M2為模板,應(yīng)用PCR方法分別擴增HV糖蛋白G1、G2的全長基因,并在基因兩端創(chuàng)建相應(yīng)的酶切位點,將PCR產(chǎn)物和表達載體pCAGGS/MCS分別雙酶切后連接,轉(zhuǎn)化大腸桿菌DH5α,經(jīng)氨芐篩選,酶切和測序鑒定正確后,兩種載體分別命名為pCAGGS-G1、pCAGGS-G2,然后將兩種糖蛋白載體共轉(zhuǎn)染Vero E6細胞,酸性MEM處理、Giemsa染色后觀察細胞融合現(xiàn)象的產(chǎn)生。同時用pCAGGS-NP與兩種糖蛋白載體共表達,看核蛋白是否對糖蛋白的融合有增強作用,并以間接免疫熒光實驗(indirect immunofluorescence assay,IIFA)來觀察糖蛋白和核蛋白的表達情況。利用基因定點突變的方法構(gòu)建了五個糖蛋白突變體,即將G1、G2上的的天冬酰胺置換為丙氨酸,根據(jù)被替換的位置,突變體分別命名為N134A、N235A、N347A、N399A、N928A。 結(jié)果1.構(gòu)建了含有包膜糖蛋白基因G1、G2的真核表達載體pCAGGS-G1和pCAGGS-G2,雙酶切鑒定目的片段分別為2.0kb和1.5kb,載體片段為4.7kb并測序證實。 2.間接免疫熒光試驗顯示糖蛋白組、核蛋白組及兩者的共表達組皆有亮綠色熒光信號產(chǎn)生,呈胞漿分布。 3.糖蛋白G1、G2共轉(zhuǎn)染后在偏酸性條件下可引起Vero E6細胞發(fā)生融合,而轉(zhuǎn)染pCAGGS-NP的細胞則沒有融合現(xiàn)象發(fā)生;將核蛋白與糖蛋白共表達后也未出現(xiàn)明顯的促進效應(yīng)。 4.構(gòu)建了五個N-連糖基化位點的突變體,測序圖譜顯示原序列中的天冬酰胺(N)均被置換為丙氨酸(A)。 結(jié)論成功構(gòu)建了漢坦病毒包膜糖蛋白基因G1、G2的真核表達載體并在Vero E6細胞中有效表達,二者的共表達可以產(chǎn)生良好的生物學(xué)活性,在酸性條件下引起Vero E6細胞發(fā)生融合。為進一步研究糖蛋白的結(jié)構(gòu)與功能,制備有效的亞單位疫苗奠定了堅實基礎(chǔ)。成功構(gòu)建了各糖基化位點的突變體,為進一步研究N-連糖基化的缺失對細胞融合的影響提供了必要條件。
[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 偽. 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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R373
本文編號：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 偽. 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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R373
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