Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.
Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.
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通过复制和非复制载体在植物中快速瞬时生产产生高质量的功能性抗 HIV 抗体。
DOI：
10.1371/journal.pone.0013976
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发表时间：
2010-11-12
期刊：
影响因子：
3.7
通讯作者：
Lomonossoff GP
中科院分区：
文献类型：
--
作者：
Sainsbury F;Sack M;Stadlmann J;Quendler H;Fischer R;Lomonossoff GP
植物和植物细胞产生大量重组蛋白的能力已经得到了很好的证实。由于在速度和产量方面的优势，最近人们的注意力转向使用瞬时表达系统，包括病毒载体，以生产在植物中具有药用价值的蛋白质。然而，病毒载体的高水平表达及其对宿主细胞的伴随作用可能会影响重组产品的质量。为了评估在植物中瞬时高水平表达的抗体的质量，我们使用基于豌豆花叶病毒(CPMV)RNA-2缺失版本的复制和非复制系统表达并表征了人类抗HIV单抗2G12。当使用非复制的CPMV-HT系统并将抗体保留在内质网(ER)中时，亲和纯化的2G12产量最高(约100 mg/kg湿重叶片组织)。糖链的质谱分析表明，糖基化模式完全由抗体是否保留在内质网中决定，而不取决于是否使用复制或非复制系统。对所有从植物中纯化的2G12变异体的结合和中和特性的表征表明，这些变异体与中国仓鼠卵巢(CHO)细胞产生的2G12的结合和中和特性大致相似。总体而言，结果表明，复制和非复制的CPMV载体能够指导产生与CHO产生的对照相似的重组免疫球蛋白。因此，无论是通过高水平表达还是病毒复制，都可以生产出复杂的重组蛋白，而对其生化性质没有明显影响。具有优良生化特性的重组药物可以在植物中瞬时生产，这使得基于CPMV的表达载体成为生物制药开发和生产的一个有吸引力的选择。
The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product. To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV) RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue) of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER). Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO) cell-produced 2G12. Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for biopharmaceutical development and production.
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影响因子：
5.4
作者：
Carette, JE;van Lent, J;van Kammen, A
通讯作者：
van Kammen, A
影响因子：
4
作者：
Ca単izares, MC;Nicholson, L;Lomonossoff, GP
通讯作者：
Lomonossoff, GP
DOI：
10.1073/pnas.96.24.13910
发表时间：
1999-11-23
影响因子：
11.1
作者：
Drake, JW;Holland, JJ
通讯作者：
Holland, JJ
DOI：
10.1073/pnas.0606631103
发表时间：
2006-10-03
影响因子：
11.1
作者：
Giritch, Anatoli;Marillonnet, Sylvestre;Gleba, Yuri
通讯作者：
Gleba, Yuri
影响因子：
56.9
作者：
HOLLAND, J;SPINDLER, K;VANDEPOL, S
通讯作者：
VANDEPOL, S