Influence of promoter and signal peptide on the expression and secretion of recombinant porcine LH extracellular domain in baculovirus/lepidopteran cells or the caterpillar system.
Influence of promoter and signal peptide on the expression and secretion of recombinant porcine LH extracellular domain in baculovirus/lepidopteran cells or the caterpillar system.
复制标题
启动子和信号肽对杆状病毒/鳞翅目细胞或毛虫系统中重组猪LH胞外结构域表达和分泌的影响。
DOI：
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发表时间：
1995
影响因子：
3.5
通讯作者：
R. Salesse
中科院分区：
文献类型：
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作者：
V. Bozon;J. Remy;E. Pajot;L. Couture;G. Biache;M. Severini;R. Salesse
猪LH受体（pLHR）胞外域的过表达已实现使用杆状病毒-昆虫细胞系统，但主要是在聚集的形式，没有分泌。为了实现这一点，选择新的杆状病毒在昆虫Sf 9细胞和毛虫中产生pLHR胞外域。在pLHR-P10-297和pLHR-mel-319杆状病毒中，pLHR cDNA分别受P10启动子和多角体蛋白基因启动子的控制。构建体含有猪信号肽（pLHR-P10-297）或蜂毒素的昆虫信号肽（pLHR-mel-319）。感染细胞在感染后3天产生1 × 10（5）-3 × 10（5）受体/细胞。产生的重组LH受体胞外域以生物活性形式分泌，并以高亲和力结合激素。感染后的毛虫比昆虫细胞产生更多的pLHR胞外域。然而，产物不分泌到血淋巴中。因此，启动子和/或信号肽修饰使得pLHR重组胞外域能够使用杆状病毒-鳞翅目细胞系统以生物活性形式分泌。此外，中等水平的表达似乎允许生物活性胞外域的产生。
Overexpression of the porcine LH receptor (pLHR) ectodomain has been achieved using the baculovirus-insect cell system but mostly in an aggregated form with no secretion. In order to carry this out, new baculoviruses were selected to produce the pLHR ectodomain in insect Sf9 cells and caterpillars. In pLHR-P10-297 and pLHR-mel-319 baculoviruses, pLHR cDNA was under the control of the P10 promoter and the polyhedrin gene promoter respectively. The constructs contained either the porcine signal peptide (pLHR-P10-297) or the insect signal peptide of melittin (pLHR-mel-319). Infected cells produced 1 x 10(5)-3 x 10(5) receptors/cell 3 days after infection. The recombinant LH receptor ectodomains produced were secreted in a biologically active form and bound the hormone with high affinity. Infected caterpillars produced a larger amount of active pLHR ectodomain that insect cells. The products were not secreted into the haemolymph however. Promoter and/or signal peptide modifications therefore enabled pLHR recombinant ectodomain secretion in a biologically active form, using the baculovirus-lepidopteran cell system. Moreover, moderate levels of expression seem to allow the production of biologically active ectodomain.