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百日咳毒素S1亚单位缺失分子的构建及其在重组杆状病毒中的表达 |
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李庆雷 于康震 马丽英 王孝铭
摘 要：目的：为探明羧基端疏水区对百日咳毒素S1亚单位分泌性的影响，试图构建缺失羧基端疏水区的S1亚单位。方法：应用PCR技术构建了6种不同信号序列修饰的缺失羧基端疏水区的S1亚单位编码基因，并使用重组杆状病毒(rBV)技术实现了这些缺失S1亚单位(tS1)在昆虫细胞中的高水平表达。结果：这些缺失S1亚单位的表达量为每万个昆虫细胞1.01～2.25 μg。细菌信号肽和流感病毒HA信号肽均能完整表达和正确剪切，但HA信号肽在昆虫细胞中的处理效率更高。结论：tS1亚单位缺失羧基端疏水区后在昆虫细胞中的表达量明显提高，这些tS1亚单位的异源性表达是由于信号肽剪切不全所导致，tS1分子表观分子量的漂移并不意味着信号肽剪切不正确。
关键词：百日咳毒素 S1亚单位 重组杆状病毒 信号肽 疏水区
分类号：R378.42
Expression of truncated S1 subunit of pertussis toxin in recombinant baculoviruses
LI Qing-Lei
Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150001
YU Kang-Zhen
Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150001
MA Li-Ying
Department of Pathophysiology of Harbin Medical University,Harbin 150086
WANG Xiao-Ming
Department of Pathophysiology of Harbin Medical University,Harbin 150086
Abstract：Objective：In order to investigate the effect of the hydrophobic domains on secretion of the subunits,we deleted the hydrophobic domains from the S1 subunits.Methods:Six signal modified S1 encoding DNA fragmants were constructed that lacked C-terminal hydrophobic sequences using PCR technique expecting to get secreted S1 proteins.Results:Those truncated S1(tS1)subunits were abundantly expressed by recombinant baculoviruses in insect cells (expression levels ranged from 1.01 to 2.25 μg/104 cells).The HA signal could be processed more efficiently than the native one,although they both could be cleaved correctly.Conclusion:The expression levels of the tS1 subunits are increased significantly after the deletion of the hydrophobic domains.The heterologous expression of the tS1 proteins is the result of incomplete signal cleavage,and the apparent MW shifting does not imply incorrect signal cleavage.
Key words：Pertussis toxin S1 subunit Recombinant baculoviruse Signal sequence Hydrophobic domain▲
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