百日咳毒素S1亚单位的分子修饰及在重组杆状病毒中的表达

Expression of genetically altered S1 subunits of pertussis toxin in recombinant baculoviruses

目的：百日咳毒素既是百日咳杆菌的主要毒性因子，又是重要的保护性抗原，其Ｓ１亚单位具有ＡＤＰ核糖转移酶活性和免疫保护性决定簇。为高效表达前构建的一个丧失酶活性但仍保持保护决定簇的Ｓ１变异子，开展了本研究。方法：通过添加人流感病毒血凝素基因信号序列或嵌膜序列的方法，对天然和变异Ｓ１亚单位基因片段作了遗传学修饰。然后使用重组杆状病毒技术使这些Ｓ１亚单位在昆虫细胞和昆虫幼虫中实现了高水平表达。结果：这些重组Ｓ１的表达水平介于每万个昆虫卵细胞０１８～１９３μｇ或每只昆虫幼虫０４６～４９８ｍｇ之间。天然信号肽和ＨＡ信号肽均可完整表达，但后者的切割效率（６１％～８１％）比前者（１６％～１９％）更高。所有带信号肽的Ｓ１亚单位均呈异质性表达（双带）。结论：重组Ｓ１亚单位的表达是高效和正确的，表达的异质性是由于信号肽切割不完全的缘故。

Objective:Pertusis toxin(PT)is both a major virulence factor of bordetella pertussis and an important immunoprotective antigen against whooping cough.The S1 subunit of PT possesses ADP ribosyltransferase activity as well as epitopes that induce protective immunity.The aim of the present research is to produce the S1 mutant abundantly which is in lack of the enzyme activity constructed previously.Methods:Have modified both native and mutant S1 subunit encoding DNA fragments by the addition of the native S1 signal sequence or the signal sequence of human influenza virus hemagglutinin(HA) gene.The modified S1 subunits were abundantly expressed by recombinant autographa californica nuclear polyhedrosis viruses in insect cells and larva.Results:The expression levels ranged from 0.18～1.93 μg/10 4 spodoptera frugiperda ovarian cells,or 0.46～4.98 mg/larva of spodoptera exigua.The native and HA signal sequences were both expressed intact,although the HA signal was processed more efficiently(61%～81%) tnan the native one(16%～19%).All S1 with signals produced two bands.Conclusion:All recombinant S1 subunits were produced correctly to high levels.The heterologous expression of rS1 proteins is the result of incomplete signal cleavage.