Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64.
Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64.
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DOI：
10.1046/j.1432-1033.2002.03135.x
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发表时间：
2002-09
期刊：
影响因子：
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通讯作者：
A. Spenger;R. Grabherr;Lars Töllner;H. Katinger;W. Ernst
中科院分区：
文献类型：
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作者：
A. Spenger;R. Grabherr;Lars Töllner;H. Katinger;W. Ernst
苜蓿银纹夜蛾核型多角体病毒（Autographa californica nuclear polyhedrosis virus）的囊膜蛋白gp 64是病毒进入昆虫细胞所必需的，因为该糖蛋白既介导pH依赖性膜融合又与宿主细胞受体结合。利用gp 64基因工程对杆状病毒颗粒进行表面修饰已经被各种策略所证实，因此已经成为分子生物学中一种重要而有力的工具。为了进一步改善杆状病毒颗粒表面上肽的呈递，通过其理论最大表面概率选择gp 64包膜蛋白内的几个插入位点，并研究有效的肽呈递。将HIV-1 gp 41的ELDKWA肽（对人mAb 2F 5具有特异性）插入糖蛋白gp 64的17个不同位置。在13例中病毒增殖成功，在4个位置的诱变没有导致完整的病毒颗粒的生产。Western印迹、FACS分析和ELISA用于表征突变体的不同结合特性。将该肽插入到天然包膜蛋白中导致杆状病毒颗粒表面上的高亲合力展示。这种方法提供了在宿主范围特异性和抗原展示方面有效修饰表面性质的可能性。
The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pH-dependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV-1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display.