Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells
Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells
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哺乳动物细胞中单纯疱疹病毒 1 型糖蛋白 gB 的细胞相关和分泌形式的表达
作者：
C. Pachl;R. Burke;L. Stuve;L. Sanchez;G. Van Nest;F. Masiarz;D. Dina
在中国仓鼠卵巢细胞系中表达了单纯疱疹病毒1型Patton株糖蛋白GB1基因。将含有二氢叶酸还原酶(DHFR)基因的表达载体和完整的GB1基因或缺失GB1基因194个羧基末端氨基酸的截短基因导入CHO DHFR缺陷细胞。放射免疫沉淀法证实，完整的GB1蛋白在CHO细胞中的表达是与细胞相关的，而截短的蛋白是由于GB1的跨膜区和C末端区域的缺失而从细胞中分泌出来的。对表达截短的GB1蛋白的细胞进行分步甲氨蝶呤筛选，获得一株GB1基因拷贝数扩增10倍、表达水平提高60倍以上的细胞系。从扩增的细胞系条件培养上清液中纯化截短的GB1蛋白。对该纯化蛋白的N-末端氨基酸序列分析确定了信号肽的切割位置，并预测了从初级蛋白中切割出一个30个氨基酸的信号序列。截短的GB1蛋白在小鼠体内也进行了免疫原性测试，并观察到高水平的抗体和对病毒攻击的保护作用。
The gene for glycoprotein gB1 of herpes simplex virus type 1 strain Patton was expressed in stable Chinese hamster ovary cell lines. Expression vectors containing the dihydrofolate reductase (dhfr) cDNA plus the complete gB1 gene or a truncated gene lacking the 194 carboxyl-terminal amino acids of gB1 were transfected into CHO DHFR-deficient cells. Radioimmunoprecipitation demonstrated that the complete gB1 protein expressed in CHO cell lines was cell associated, whereas the truncated protein was secreted from the cells due to deletion of the transmembrane and C-terminal domains of gB1. Cells expressing the truncated gB1 protein were subjected to stepwise methotrexate selection, and a cell line was isolated in which the gB1 gene copy number had been amplified 10-fold and the level of expression of gB1 had increased over 60-fold. The truncated gB1 protein was purified from medium conditioned by the amplified cell line. N-terminal amino acid sequence analysis of this purified protein identified the signal peptide cleavage site and predicted the cleavage of a 30-amino-acid signal sequence from the primary protein. The immunogenicity of the truncated gB1 protein was also tested in mice, and high levels of antibody and protection from virus challenge were observed.
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DOI：
--
发表时间：
1983
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
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DOI：
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期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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DOI：
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发表时间：
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期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
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作者：
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DOI：
--
发表时间：
1986
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
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作者：
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